An engineered exosome sphere delivery system loaded with therapeutic drugs, preparation method and application thereof

Abstract

The present application relates to the technical field of medicine, and relates to an engineered exosome ball delivery system loaded with a therapeutic drug and a preparation method and application thereof. The system is a platelet exosome loaded with a therapeutic drug, and then the surface phosphatidylserine of the drug-loaded exosome is shielded, that is, the exosome ball delivery system is obtained. Compared with the exosome, the exosome ball has stronger resistance to macrophage phagocytosis, longer blood circulation time, and better tumor penetration ability than the platelet. In addition, among the tested preparations, the exosome ball shows higher uptake rate of tumor cells and produces the strongest tumor inhibition effect, and can well inhibit tumor growth and eliminate circulating tumor cells. These findings show that the exosome ball solves the limitations of the biological carrier system and provides great potential for efficient and precise drug delivery.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and specifically to an engineered exosome delivery system for loading therapeutic drugs, its preparation method, and its application. Background Technology

[0002] Cells and their derived exosomes are widely used as biological carrier systems. Cells exhibit superior targeting specificity and prolonged circulation due to their rich array of surface proteins, while exosomes, due to their small size, can cross barriers and effectively penetrate tumors. However, challenges remain: the large size of cells limits tissue penetration, and exosomes have limited targeting accuracy and short circulation times.

[0003] Exosomes, as natural nanoscale vesicles, have attracted much attention in the field of chemotherapy drug delivery due to their unique biological characteristics. Their advantages include high biocompatibility, low immunogenicity, natural targeting, and the ability to cross biological barriers. However, exosomes face significant challenges in clinical translation, with short blood circulation time (typically only minutes to hours) being a key bottleneck. The main reason for this short blood circulation time is the rapid clearance by the mononuclear phagocytic system (MPS). Phosphatidylserine residues or specific proteins, such as low expression of CD47, exposed on the surface of exosomes can be recognized by liver / spleen macrophages, triggering phagocytic clearance through "eat me" signals. Summary of the Invention

[0004] The technical problem solved by this invention is to overcome the shortcomings of existing technologies, and to provide an engineered long-acting exosome sphere delivery system for loading therapeutic drugs, along with its preparation method and application, taking into account the long-circulation effect of exosomes and cells and their tumor targeting capabilities.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] An engineered exosomal sphere delivery system for loading therapeutic drugs is disclosed. The system consists of platelet exosomes encapsulating therapeutic drugs, followed by shielding the surface of the drug-loaded exosomes from phosphatidylserine.

[0007] The system consists of platelet exosomes encapsulating therapeutic drugs, then shielding the surface of the drug-loaded exosomes from phosphatidylserine, and finally connecting the individual exosome spheres via pH-sensitive bonds.

[0008] The therapeutic drug is one or more of the anti-tumor drugs.

[0009] The antitumor drugs mentioned are small molecule drugs, such as doxorubicin, paclitaxel, cisplatin, SN38, methotrexate and their derivatives.

[0010] The surface phosphatidylserine of the drug-loaded exosomes is shielded by co-incubating the platelet exosomes carrying therapeutic drugs with connexin V, thereby achieving the shielding of their surface phosphatidylserine.

[0011] A method for preparing the engineered exosome delivery system for loading therapeutic drugs, as described above.

[0012] (1) Extraction and purification of platelet exosomes;

[0013] (2) Preparation of exosomes loaded with therapeutic drugs;

[0014] (3) Shielding the surface phosphatidylserine of drug-loaded exosomes to obtain an engineered exosome delivery system for internally loaded therapeutic drugs.

[0015] After shielding the surface phosphatidylserine of the drug-loaded exosomes in step (3), they are connected into spheres by pH-sensitive bonds, thus obtaining an engineered exosome sphere delivery system for loading therapeutic drugs.

[0016] To elaborate further,

[0017] (1) Extraction and purification of platelet exosomes;

[0018] (2) Prepare exosomes loaded with therapeutic drugs; dissolve the therapeutic drugs and incubate them with the extracted platelet exosomes, then centrifuge to remove the free therapeutic drugs to obtain platelet exosomes loaded with therapeutic drugs for later use.

[0019] (3) Shield the surface phosphatidylserine of drug-loaded exosomes; take the platelet exosomes loaded with therapeutic drugs from step (2) and incubate them with connexin V, centrifuge, and remove unconnected connexin V.

[0020] After shielding the surface phosphatidylserine of the drug-loaded exosomes in step (3), the exosomes are connected into spheres by connecting one end of the pH-sensitive bond MAL-PEOz-COOH to the thiol group on the surface of the exosomes and the other end to the amino group.

[0021] The above-mentioned extraction and purification of platelet exosomes involves taking anticoagulated fresh blood, obtaining platelets through gradient centrifugation, washing and purifying them, dissolving the platelets in HEPES solution, adding A23187 to promote exosome secretion, and then performing differential centrifugation to obtain platelet exosomes.

[0022] The platelets mentioned are murine in origin and are extracted from fresh mouse blood plasma.

[0023] Application of the engineered exosome sphere delivery system for loading therapeutic drugs, and application of the engineered exosome sphere delivery system for loading therapeutic drugs in drug delivery systems.

[0024] The application of the engineered exosome sphere delivery system for loading therapeutic drugs, and the application of the engineered exosome sphere delivery system for loading therapeutic drugs in anti-tumor growth and metastasis.

[0025] The engineered exosomes loaded with therapeutic drugs were prepared and their properties were investigated. Taking doxorubicin as an example, platelet exosomes loaded with doxorubicin (PexD), platelet exosomes loaded with doxorubicin and shielded by phosphatidylserine (PPexD), and engineered platelet exosomes loaded with doxorubicin (SPPexD) were prepared. The properties of each substance were then evaluated.

[0026] 1) The physicochemical properties of the prepared platelet exosomes loaded with therapeutic drugs were characterized, such as the morphology and structure of engineered exosomes, protein expression, drug release, and storage stability.

[0027] 2) The adhesion, uptake, and cytotoxicity of platelet exosomes carrying therapeutic drugs were investigated at the cellular level.

[0028] 3) Pharmacokinetic behavior and in vivo distribution of platelet exosomes loaded with therapeutic drugs.

[0029] 4) The ability of platelet exosomes loaded with therapeutic drugs to inhibit tumor growth and metastasis.

[0030] Advantages of this invention:

[0031] The drug delivery system of this invention is highly efficient and low in toxicity, prolongs the blood circulation time of platelet exosomes, and exhibits superior tumor penetration ability. Specifically:

[0032] 1. The preparation of the engineered exosome sphere delivery system of the present invention includes extracting and purifying platelet exosomes, loading chemotherapeutic drugs into the platelet exosomes, shielding the surface of the drug-loaded exosomes with phosphatidylserine, and connecting the drug-loaded exosomes with phosphatidylserine shielding into spheres via pH-sensitive bonds. The engineered exosome sphere delivery system of the present invention can achieve stable in vitro storage and long-term in vivo circulation. Furthermore, the engineered long-acting platelet exosome sphere delivery system of the present invention, through the highly efficient tumor targeting of platelet exosomes, can achieve precise targeting of tumor cells and circulating tumor cells, thereby releasing cytotoxic chemotherapeutic drugs and effectively inhibiting tumor growth and metastasis.

[0033] 2. This invention relates to a drug delivery system based on engineered platelet exosome spheres. This system aims to enhance drug targeting capabilities while increasing drug circulation time, maximizing the clinical application potential of platelet exosome-related formulations. This invention contains a bioengineered platelet exosome sphere system carrying doxorubicin. Due to its specific tumor targeting and capture capabilities, this system can specifically deliver chemotherapeutic drugs to the tumor site to inhibit tumor growth and metastasis. Upon reaching the tumor site, under the pH regulation of the tumor site, the exosome spheres disintegrate into individual exosomes, thereby enhancing tumor penetration and achieving a therapeutic effect.

[0034] In summary, the platelet exosomes containing therapeutic drugs prepared in this invention can achieve stable in vitro storage and long-term in vivo circulation while targeting tumor cells and circulating tumor cells at the tumor site. Attached Figure Description

[0035] Figure 1 A schematic diagram illustrating platelet exosome extraction and SPPexD preparation;

[0036] Figure 2 Morphological characterization of SPPexD;

[0037] A is a transmission electron microscope image of SPPexD; B is an appearance image of SPPexD and other formulations; C is the flow cytometry result of phosphatidylserine detection in SPPexD and other formulations.

[0038] Figure 3 For the formulation characterization of SPPexD;

[0039] A shows protein analysis using SDS-PAGE; B shows the results of Western blot analysis; C shows the stability assessment of SPPex and platelets; D shows the DOX release curves of different formulations in PBS at pH 7.4; E shows the DOX release curves of different formulations in PBS at pH 6.5; F shows the particle size change of SPPexD after storage at 4°C for one week.

[0040] Figure 4 To investigate the anti-phagocytic properties and tumor cell adhesion of SPPexD;

[0041] A: Various DiR-labeled SPPex and other vectors were incubated with RAW264.7 macrophages at 37°C for 1 hour to assess their phagocytic activity; B: DiR-labeled SPPex and other vectors were incubated with B16-F10 cells at 4°C for 1 hour to assess their adhesion ability.

[0042] Figure 5 The therapeutic effect of SPPexD at the cellular level;

[0043] A. Flow cytometry analysis of B16-F10 cell uptake after incubation with the corresponding agents for 0.5 and 2 hours; B. Quantitative analysis of cell uptake (G1: Dox, G2: PexD, G3: PPexD, G4: Platelet-Dox, G5: SPPexD); C. Evaluation of the cytotoxicity of various doxorubicin-containing agents on B16F10 cells; D. HMGB1 release after 24 hours of treatment with various doxorubicin-containing agents on B16-F10 cells; E. ATP release after 24 hours of treatment with the same agents on B16-F10 cells; F. Dendritic cell maturation induced by various agents.

[0044] Figure 6 To investigate the tumor sphere penetration of SPPexD and other formulations; Pex(G1), PPex(G2), Platelet(G3), SPPexD(G4).

[0045] Figure 7 SPPexD capture of circulating tumor cells.

[0046] Figure 8 Blood circulation time of different formulations.

[0047] Figure 9 To investigate the tissue distribution and antitumor effects of SPPexD;

[0048] A. In vivo fluorescence imaging of B16-F10 tumor-bearing mice at 1, 6, and 12 hours after intravenous injection of free DiR solution, DiR-labeled Pex, PPex, platelet mimics, and SPPex; B. In vitro fluorescence imaging of major tissues collected at 1, 6, and 12 hours after injection of the formulation; C. Tumor inhibition effect of SPPexD; D. Survival rate of mice in each treatment group; E. Body weight changes of mice in different treatment groups; F. Quantification of tumor weight in mice after treatment; G. Tumor growth curves of mice in different groups during treatment (G1: Control; G2: Pex; G3: PPex; G4: Platelet; G5: SPPex; G6: DOX; G7: PexD; G8: PPexD; G9: Platelet-DOX; G10: SPPexD); H. Assessment of liver and kidney function in mice treated with DOX-containing formulations. Detailed Implementation

[0049] 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.

[0050] This invention relates to an exosome spheroid delivery system that combines phosphatidylserine shielded by conjugation-conjugation-V with platelet-derived exosomes linked by a pH-sensitive MAL-PEOz-COOH bond for drug delivery applications. Studies have shown that, compared to exosomes, exosome spheroids improve blood circulation by upregulating CD47 expression and shielding phosphatidylserine, thereby minimizing immune clearance. Furthermore, increased P-selectin expression promotes adhesion to circulating tumor cells, thus improving targeting efficiency. Upon reaching the tumor site, the hydrazone bonds of the exosome spheroids are protonated in the acidic tumor microenvironment, leading to their disintegration into uniformly sized exosomes that penetrate deeper into the tumor compared to platelets. These findings indicate that exosome spheroids address these challenges and offer significant potential for efficient and precise drug delivery.

[0051] Example 1: Preparation and characterization of engineered platelet exosomes loaded with therapeutic drugs.

[0052] Preparation and characterization of SPPexD

[0053] Exosomes (Pex) were extracted from platelets using the classic differential centrifugation method.

[0054] Mix 500 micrograms of exosomes with 500 micrograms of DOX at a mass ratio of 1:1, incubate at 37 degrees Celsius for 1 hour, and then centrifuge at 100,000g. The precipitate is the DOX-loaded platelet-derived exosome (PexD).

[0055] 1 mg of PexD was incubated with 50 μg of annexin V for 1 hour, followed by centrifugation at 100,000 g to obtain PS-masked platelet-derived exosomes modified with annexin V (PPexD). 1 mg of PPexD was then incubated with 25 μg of MAL-PEOz-COOH overnight at 37°C to obtain PS-masked exosomes loaded with DOX (SPPexD). Figure 1 ).

[0056] Scanning electron microscope (SEM) Figure 2 A) The successful fabrication of spherical SPPexDs with an average size of 350 nm was confirmed. Images of the SPPexDs and their appearance from each step of the fabrication process were also captured. Figure 2 B). Flow cytometry showed that the fluorescence intensity of phosphatidylserine (PS) on PPexD was significantly lower than that on normal PexD, similar to that on resting platelets. These findings suggest that PPexD can effectively mask PS on the surface of exosomes, potentially preventing macrophage recognition (B). Figure 2 C).

[0057] SDS-PAGE analysis showed that SPPexD retained the unique proteins expressed on platelets and Pex. Figure 3 A). Western blotting confirmed the expression of essential proteins on platelets, Pex, and SPPexD. Figure 3 (B) Notably, the exosome biomarker CD63 was expressed on SPPexD, and also on purified small extracellular vesicles obtained by differential ultracentrifugation. The cell adhesion molecule P-selectin was identified on SPPexD, which can bind to CD44 expressed on B16-F10 cells. Similarly, CD47 was detected on SPPexD, which interacts with Sirpα on the surface of macrophages, transmitting inhibitory signals and preventing phagocytosis. These proteins coexisted on platelets, Pex, PPexD, and SPPexD, indicating that functional proteins were retained on SPPexD during modification.

[0058] Simultaneously, the cumulative release of DOX from the products obtained during the above preparation process was detected. Specifically, different formulations were encapsulated in dialysis bags, and then the release was measured in PBS under physiological conditions (pH 7.4). Figure 3 (D) As shown in the figure, the cumulative release of DOX from PexD reached 45.3% at 8 hours and increased to 60.8% at 24 hours. PPexD exhibited similar release behavior to PexD. The results indicate that annexin V modification did not significantly alter the DOX release kinetics. However, the release rate of SPPexD decreased, with a cumulative DOX release of 20.2% at 8 hours and 46.2% at 24 hours. This phenomenon may be due to the spherical structure of exosomes creating an additional diffusion barrier, hindering DOX release.

[0059] The products obtained in the above preparation process were subjected to appearance photography and particle size determination. Figure 3 E, Figure 3 As shown in Figure F, SPPexD can be stably stored at 4°C for one week, while platelets showed aggregation after one day of storage at 4°C. This indicates that SPPex has better stability than platelets, thus ensuring the efficacy and safety of the drug.

[0060] Example 2: Effects of SPPexD on macrophages and tumor cells at the in vitro cellular level.

[0061] Because of the large number of macrophages in the liver and spleen, exosomes tend to accumulate mainly in these organs.

[0062] To evaluate the antiphagocytic ability of SPPexD, we used DIR-labeled SPPex (specifically, phosphatidylserine-modified exosomes (PPex) were obtained by incubating 1 mg of platelet exosomes (Pex) with 50 μg of membrane-connecting protein V for 1 h, and then incubating PPex with 25 μg of MAL-PEO2-COOH overnight to obtain SPPex; all steps were performed at 37°C) to reduce DOX fluorescence interference. Subsequently, DIR-labeled Pex, PPex, platelets, and SPPex were incubated with macrophages for 1 hour (1 mg of DIR and 1 mg of the carrier were incubated at 37°C for 30 minutes, followed by centrifugation to obtain different DIR-labeled formulations). Figure 4 As shown in Figure A, the DIR-labeled platelet group exhibited the lowest macrophage uptake, likely due to low PS content and high CD47 expression in resting platelets. Both the PPex and SPPex groups showed enhanced antiphagocytic capacity compared to the single exosome group. Notably, SPPex exhibited the strongest antiphagocytic effect due to exosome aggregation. This effect is attributed to the shielding effect of PS and the increased number of CD47 sites (the recognition site for macrophage Sirpα), providing a basis for the prolonged blood circulation of SPPexD.

[0063] To assess the cell adhesion properties of exosome spheroids, B16-F10 cells were incubated with fluorescently labeled Pex, PPex, SPPex, or platelets at 4°C for 1 hour. Figure 4 As shown in Figure B, Pex and PPex exhibited comparable fluorescence intensities, indicating that modifying the exosome surface with annexin V did not alter the adhesion of Pex to tumor cells. However, in B16-F10 cells, DIR-labeled SPPex and platelets showed higher fluorescence intensities than Pex, suggesting that exosome aggregation into spheroids improved their adhesion to tumor cells. This increased adhesion was attributed to increased exposure of P-selectin (the recognition site of CD44) on the surface of the exosome spheroids.

[0064] Flow cytometry was used to quantitatively assess the uptake of DOX by B16-F10 cells in different solutions. Specifically, 5000 B16-F10 cells were seeded per well in a 24-well plate and incubated overnight. The cells were then incubated at 37°C for 0.5 hours and 2 hours, respectively, with different exosomes prepared according to Example 1 containing 3 μg of DOX. The cells were then transferred to flow cytometry tubes for analysis; DOX and DOX-loaded platelets (Platelet-Dox) were used as controls.

[0065] like Figure 5As shown in A and 5B, SPPexD exhibits a superior ability to enhance DOX uptake by B16-F10 cells compared to DOX-loaded platelets. Although platelets adhere to B16-F10 cells better than SPPexD, B16-F10 cells are unable to effectively internalize larger platelets. DOX can only passively diffuse from platelets into cells. In contrast, SPPexD can not only effectively adhere to tumor cells but also be actively internalized by B16-F10 cells.

[0066] The in vitro cytotoxicity of different DOX-containing formulations was evaluated using MTT assay. Specifically, B16F10 cells were seeded at a density of 1,500 cells per well in 96-well plates and cultured overnight. Cells were then treated with various agents and incubated for 24 hours. MTT solution was then added to the plates and incubated at 37°C for 4 hours. After removing the MTT solution, 100 μL of dimethyl sulfoxide (DMSO) was added, and the absorbance at 570 nm was measured using a microplate spectrophotometer.

[0067] like Figure 5 As shown in Figure C, the IC50 values ​​of SPPexD, PexD, and DOX-loaded platelets were 0.094, 0.184, and 0.307 μg / mL, respectively. The SPPexD group showed enhanced cellular uptake of DOX, resulting in SPPexD having a significantly higher cytotoxic effect on B16-F10 cells compared to other formulations.

[0068] Chemotherapy drugs, such as DOX, can induce immunogenic cell death (ICD) in tumor cells, thereby triggering the maturation of dendritic cells (DCs). Specifically, after treating B16-F10 cancer cells at 37°C for 24 hours with SPPexD, PPexD, DOX-loaded platelets (Platelet-Dox), PexD, and free DOX (3 μg DOX), the release of HMGB1 and ATP was measured using a kit. Figure 5 D, E). The blank group consisted of untreated tumor cells.

[0069] The results showed that, compared with other treatments, SPPexD led to higher CRT expression and greater release of HMGB1 and ATP, key signaling molecules involved in ICD. Furthermore, co-culturing bone marrow-derived dendritic cells (BMDCs) with SPPexD-treated tumor cells resulted in increased expression of the co-stimulatory molecules CD80 and CD86, which are indicators of dendritic cell maturation. These findings suggest that SPPexD-induced ICD effectively promotes DC maturation. Figure 5 F).

[0070] To further evaluate the drug delivery potential of SPPexD and related drugs, multicellular spheroids (MCS) were used as an in vitro three-dimensional (3D) model simulating B16-F10 tumor tissue. This model was used to assess the permeability of SPPexD. To mitigate interference from DOX fluorescence, DIR-labeled Pex, PPex, platelets, and SPPexD were used and incubated separately with MCS at pH 6.5 for 6 hours. Figure 6 As shown, after 1 hour, DIR-labeled platelets (red) and SPPex, exhibiting strong adhesion, were effectively adsorbed onto the exterior of the MCS. Due to their small particle size, PPex and Pex could be detected inside the MCS, albeit at lower intensities. After 6 hours, the fluorescence intensity of SPPex within the MCS was comparable to that of PPex and Pex, while platelets primarily remained at the edges of the MCS. This is because the adsorbed SPPex decomposed into individual exosomes at low pH, subsequently dispersing into platelets.

[0071] Example 3: The effect of SPPexD in eliminating circulating tumor cells.

[0072] Circulating tumor cells (CTCs) originate from solid tumors, are isolated from the main tumor mass, and enter the bloodstream. In cancer patients, these CTCs may exist in the blood as single cells or clusters, showing considerable metastatic potential. To evaluate the ability of SPPexD to capture CTCs, experiments were conducted in which mice were intravenously injected with saline, DOX solution, PexD, PPexD, DOX-loaded platelets (Platelet-Dox), or SPPexD (based on an injection volume of 40 μg DOX per mouse and 100 μL of saline per mouse).

[0073] To simulate the behavior of CTCs in vivo, 2×10⁻⁶ tubes were inserted via the tail vein. 6 Single-cell suspensions of B16F10 tumor cells were injected into C57BL / 6 mice in each of the above groups.

[0074] like Figure 7As shown, mice treated with SPPexD exhibited almost no metastatic nodules in their lungs, highlighting the superior efficiency of SPPexD in capturing CTCs. While PexD and DOX-loaded platelet groups also showed some anti-metastatic activity, their efficacy was significantly lower compared to SPPexD. Compared to other treatment groups, SPPexD demonstrated superior efficacy in inhibiting tumor metastasis, primarily due to its multiple advantages. Enhanced anti-phagocytic capacity allows it to effectively capture CTCs before immune clearance; its excellent adhesion properties enable it to bind tightly to CTCs; and its smaller particle size compared to platelets allows it to more easily cross the cell barrier and penetrate deeper. Furthermore, in the bloodstream, SPPexD exhibits higher P-selectin levels compared to exosomes, enabling it to bind more effectively to CD44 on CTCs, thereby enhancing its CTC-capturing ability. After successful CTC capture, the DOX encapsulated within SPPexD directly eliminates tumor cells, reducing the risk of metastasis.

[0075] Example 4: Tissue distribution and antitumor effect of SPPexD.

[0076] A mouse model of melanoma was established. First, pharmacokinetic studies were conducted to evaluate the anti-phagocytic properties of SPPexD. Specifically, mice were injected via tail vein with different exosomes (intravenous injection of DOX, PexD, PPexD, or SPPexD) (injection value calculated as DOX, 5 mg / kg). Blood samples were collected from the orbital sinus at different time points, and the DOX concentration was determined using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0077] like Figure 8 As shown, the DOX solution was metabolized within 10 minutes after injection, while the half-life of DOX in PexD was 0.46 hours. The half-life of DOX in PS-masked PPexD reached 1.21 hours, which was 2.6 times that of DOX in PexD, indicating that PS masking can prolong the circulation time of exosomes in the blood. The half-life of DOX in SPPexD was 2.41 hours, which was 5.24 times that of PexD and 1.99 times that of PPexD, respectively, significantly prolonging the blood circulation time of DOX.

[0078] Subsequently, biodistribution studies were conducted in the same animal model using DIR-labeled formulations (DIR, DIR-labeled Pex, DIR-labeled PPex, DIR-labeled platelets, and DIR-labeled SPPex). Figure 9A mouse model carrying B16F10 tumors was established. Seven days later, mice (n=3) received 1 mg / kg of various DiR-labeled vectors. Images were taken at 748 nm at different time points using IVIS Spectrum. Tissues were analyzed at 1, 6, and 12 hours after euthanasia.

[0079] Notably, SPPex accumulation in tumors peaked 6 hours post-injection, with SPPex levels being 2.01 times and 1.82 times higher than Pex and PPex, respectively. Furthermore, compared to Pex absorbed by macrophage-rich organs such as the liver and spleen, SPPex exhibited reduced aggregation, similar to platelets. In summary, due to its masking of phagocytic signals and enhanced targeting of P-selectin, SPPex possesses a longer circulation time, high targeting similar to platelets, and good tumor site penetration.

[0080] Furthermore, the antitumor activity of SPPexD was evaluated in subcutaneous B16F10 tumors, and the results showed that the tumor inhibition rate of SPPexD was significantly higher than that of PexD and DOX-loaded platelets. Figure 9 C, F, G). Notably, after treatment with SPPexD, tumors in mice gradually regressed, and the survival time of the mice significantly increased. Figure 9 D), 40% of the mice survived for more than 50 days. Furthermore, assessment of systemic toxicity showed that, except for SPPexD and platelet-DOX, there was only a slight decrease in body weight in the different treatment groups ( Figure 9 E). This may indicate systemic toxicity. Conversely, there was no change in body weight in mice treated with SPPexD and Platelet-DOX. HE staining results showed no significant signs of tumor metastasis in major organs in the SPPexD treatment group. Figure 9 DOX is a classic and effective anthracycline antibiotic with significant antitumor activity and is widely used to treat various malignant tumors. However, its therapeutic application is limited due to its hepatotoxicity and nephrotoxicity. Hepatotoxicity assessment ( Figure 9 H) showed that, compared with the saline group, mice treated with DOX, PexD, and PPexD had elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN). Conversely, the ALT, AST, and BUN levels in the Platelet-DOX and SPPexD treatment groups were comparable to those in the saline group. These results indicate that Platelet-DOX and SPPexD have good safety profiles, attributed to the long blood circulation time and efficient targeting ability of SPPexD. In conclusion, SPPexD, as a novel drug delivery system, can effectively deliver DOX to tumor sites while significantly reducing hepatotoxicity and nephrotoxicity, and holds promise as a new clinical treatment option.

[0081] In summary, the engineered exosome sphere system of this invention possesses long-term circulation in the bloodstream and tumor targeting capabilities, enabling targeted delivery of loaded drugs to kill tumor cells and circulating tumor cells. Upon reaching the tumor site, the exosome spheres dissociate into individual exosomes, allowing for deep penetration into the tumor and thereby killing the tumor, significantly inhibiting tumor growth and metastasis.

Claims

1. An engineered exosome delivery system for loading therapeutic drugs, characterized in that, The system consists of platelet exosomes encapsulating doxorubicin, then shielding the surface of the drug-loaded exosomes from phosphatidylserine, and using a pH-sensitive bond MAL-PEOz-COOH to connect one end of the exosome surface to a thiol group and the other end to an amino group, thus connecting the exosomes into spheres. The surface phosphatidylserine of the drug-loaded exosomes is shielded by co-incubating the platelet exosomes carrying therapeutic drugs with connexin V, thereby achieving the shielding of their surface phosphatidylserine.

2. A method for preparing an engineered exosome delivery system for loading therapeutic drugs as described in claim 1, characterized in that... (1) Extraction and purification of platelet exosomes; (2) Preparation of exosomes loaded with doxorubicin; (3) Shielding the surface phosphatidylserine of drug-loaded exosomes. The shielding of the surface phosphatidylserine of drug-loaded exosomes is achieved by co-incubating platelet exosomes carrying therapeutic drugs with membrane bond V to achieve shielding of their surface phosphatidylserine. A pH-sensitive bond MAL-PEOz-COOH is used to connect one end of the thiol group on the surface of the exosome and the other end to connect the amino group to connect the exosomes into spheres, thereby obtaining an engineered exosome sphere delivery system carrying therapeutic drugs.

3. The application of the engineered exosome delivery system for loading therapeutic drugs as described in claim 1, characterized in that, The application of the engineered exosome delivery system for loading therapeutic drugs in the preparation of drugs against melanoma growth and metastasis.

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