Mesenchymal stem cell exosome-based pH-responsive drug delivery system and preparation method thereof

Abstract

The invention provides a pH response type mesenchymal stem cell exosome drug delivery system (pH-EVs) and a preparation method thereof. According to the system, stearic acid modified histidine polypeptide (SA-His22) is introduced into an MSC-derived exosome membrane, so that the response release capability of a carrier to a tumor or inflammatory acidic microenvironment is realized. SA-His22 is embedded into an EV phospholipid bilayer through a hydrophobic effect, and histidine is protonized under an acidic condition, so that membrane structure reconstruction is initiated, and drug release is promoted. Meanwhile, the modification strategy improves the membrane stability and target cell uptake efficiency of the exosome, and is especially suitable for precise treatment of solid tumors such as pancreatic cancer. The system disclosed by the invention has good biocompatibility, drug loading capacity and pH responsiveness, and is suitable for loading delivery of various treatment drugs such as small molecule drugs and nucleic acid.

Description

Technical Field

[0001] The present invention provides a pH-responsive drug delivery system based on mesenchymal stem cell exosomes and a preparation method thereof, which has membrane potential regulation and high uptake capacity, is suitable for the precise treatment of diseases such as tumors and inflammation, and belongs to the field of biopharmaceutical technology. Background Art

[0002] Currently, research on drug delivery systems focuses on improving targeting, achieving controlled release, and reducing systemic toxicity. While traditional drug carriers such as liposomes and polymer nanoparticles have improved drug bioavailability to some extent, they still suffer from common deficiencies: unstable drug release rates, poor targeting, and nonspecific drug distribution in non-target tissues, leading to significant toxic side effects.

[0003] In recent years, mesenchymal stem cell (MSC)-derived exosomes (EVs) have emerged as promising drug delivery vehicles due to their unique natural nanostructure, high biocompatibility, low immunogenicity, and strong chemotaxis. MSC-EVs not only stably encapsulate and transport chemotherapeutic drugs, nucleic acids (such as RNA, miRNA, siRNA), proteins, and small molecules, playing a central role in intercellular communication and signaling regulation, but also inherit the beneficial properties of parent MSC cells in tumor microenvironment (TME) regulation, anti-fibrosis, and anti-inflammatory properties. Notably, MSCs have the ability to actively recruit to the TME, and EVs derived from them naturally possess the potential to target the tumor microenvironment, making them an ideal targeted delivery platform for a variety of solid tumors, including pancreatic cancer.

[0004] In the current research on engineered EVs, the membrane modification strategy of functional peptides is widely used to achieve more efficient and precise delivery performance. 22 Because of its excellent pH sensitivity due to protonation in the weakly acidic tumor environment, His was used to construct a responsive release platform. 22 It is a typical hydrophilic structure and lacks a hydrophobic domain that binds to the lipid membrane, which makes it easy to fall off when modified on the EV membrane surface and has insufficient stability, limiting its application effect in the in vivo environment.

[0005] However, existing chemotherapy drug delivery systems face numerous challenges, particularly during paclitaxel (PTX) therapy, where therapeutic efficacy is often limited by the formation of an immunosuppressive tumor microenvironment induced by paclitaxel. Paclitaxel promotes the expansion and recruitment of myeloid-derived suppressor cells (MDSCs), impairing T cell-mediated anti-tumor immune responses and thereby interfering with the effectiveness of immunotherapy strategies. Furthermore, existing pH-responsive exosome systems suffer from limitations such as nonspecific drug release, insufficient membrane stability, and poor targeting, hindering the delivery of precise and effective treatments. To address these challenges, the present invention proposes an innovative combination therapy strategy: combining the small molecule Vacuolin-1 with paclitaxel-based chemotherapy. This strategy not only inhibits autophagy mediated by tumor stromal cells (PSCs) and disrupts metabolic interactions between the tumor stroma and tumor cells, but also, through the Vacuolin-1 mechanism, reduces MDSC recruitment, reshapes the immune microenvironment, and mitigates paclitaxel-induced immunosuppression. This system effectively enhances drug targeting and therapeutic efficacy, optimizes paclitaxel efficacy, and mitigates side effects, thus providing a novel approach for precision therapy. Summary of the Invention

[0006] The purpose of the present invention is to provide a pH-responsive drug delivery system based on mesenchymal stem cell exosomes and its preparation method (abbreviated as pH-EVs). The system introduces a stearic acid-modified histidine peptide (SA-His) on the surface of MSC-derived exosomes (abbreviated as EVs) to 22 ), giving it pH-responsive release capability in a specific acidic microenvironment. 22 The histidine residues in the structure are protonated under acidic conditions, triggering the microstructural reorganization of the EV membrane (such as membrane micropores or swelling), thereby achieving controlled release of drugs at the targeted lesion site and improving the accuracy and efficiency of treatment.

[0007] The pH-EVs system provided by the present invention not only solves the technical difficulties of existing delivery platforms such as imprecise drug release, low release efficiency and unstable membrane modification, but also enhances the cellular uptake efficiency of exosomes through membrane potential regulation and structural sensitivity. It is particularly suitable for multi-target delivery and combined treatment in the complex microenvironment of solid tumors such as pancreatic cancer.

[0008] More importantly, this membrane modification strategy also significantly enhanced the uptake of EVs in target cells, especially under acidic conditions such as tumor microenvironment, where the charge change and structural adaptation transformation of the membrane surface can promote the fusion and internalization of exosomes with cell membranes. Flow cytometry and confocal microscopy imaging results showed that the SA-His 22The functionally modified exosomes show higher cell uptake rate in Panc02 and PSC cells, especially PSC cells, because they have stronger vesicle internalization capacity in the tumor microenvironment, so that the system has stronger stromal targeting delivery advantage.

[0009] The application introduces a hydrophobic stearic acid (SA) molecule at the N-terminus of His 22 , to construct an amphiphilic structural unit SA-His 22 . This structure can be stably inserted into the EV phospholipid bilayer through hydrophobic interaction, significantly improving the anchoring efficiency of the peptide segment and enhancing its membrane modification stability. At the same time, this modification strategy also endows EVs with membrane responsiveness, enabling them to undergo structural reconstruction in acidic microenvironments (such as tumor TME or intracellular lysosomes), thus achieving precise release of the drug delivery system. More importantly, SA-His 22 modification also significantly improves the cell uptake capacity of EVs in acidic environments. After protonation on the membrane surface, the surface charge and structure of the membrane are changed, promoting fusion and endocytosis with the target cell membrane.

[0010] The pH-EVs system provided by the application not only solves the technical problems of existing delivery platforms, such as inaccurate drug control, low release efficiency, and unstable membrane modification, but also enhances the cell uptake efficiency of exosomes through membrane potential regulation and structural sensitivity, especially for multi-target delivery and combination therapy in the complex microenvironment of solid tumors such as pancreatic cancer. Especially in the combined application of paclitaxel (PTX) and Vacuolin-1, the advantages of the system can significantly improve the therapeutic effect.

[0011] In addition, the application also combines small molecule Vacuolin-1, as an autophagy inhibitor, to block the fusion of autophagosomes and lysosomes, inhibit the recruitment of MDSCs, and effectively alleviate the immunosuppressive tumor microenvironment induced by paclitaxel (PTX). Vacuolin-1, when used in combination with PTX, can optimize the immune microenvironment, inhibit autophagy support mediated by tumor stromal cells (PSCs), and interfere with the metabolic interaction between the stroma and the tumor, thereby achieving more precise treatment effects. Through this drug combination, the therapeutic effect of PTX and Vacuolin-1 has been significantly improved, further enhancing the targeting delivery capability of exosomes in acidic tumor microenvironments, greatly improving the antitumor effect of paclitaxel, and reducing the immunosuppressive effect, thus providing a new solution for precise treatment.

[0012] The histidine polypeptide SA-His 22 provided by the application has the following molecular structure:

[0013] The SA-His 22It consists of two parts: one is the hydrophilic polyhistidine peptide (His 22 ), and the second is the hydrophobic stearic acid (SA) molecule.

[0014] Among them, His 22 It is a hydrophilic peptide segment and lacks the hydrophobic domain required for inserting into the lipid membrane, making it difficult to stably anchor on the surface of the biological membrane. In order to improve its membrane anchoring ability, the present invention covalently links SA to His 22 The N-terminus forms an amphiphilic structural unit, and the SA part is inserted into the lipid bilayer through hydrophobic interaction, thereby realizing His 22 Stable membrane binding effectively reduces the risk of peptide shedding during delivery.

[0015] The preparation method of SA-His22 of the present invention comprises the following steps:

[0016] 1. SA activation reaction: Dissolve octadecanoic acid and an equimolar amount of N-hydroxysuccinimide (NHS) in anhydrous dimethylformamide (DMF) and react with stirring at room temperature for 4–8 hours to generate the active ester SA-NHS.

[0017] 2.His 22 Coupling reaction: His 22 Dissolve in a small amount of buffer, slowly add SA-NHS solution, and continue to react at room temperature or under appropriate conditions for several hours to achieve directional coupling of the N-terminal amino group.

[0018] 3. Purification step: After the reaction is completed, dialyze in deionized water for 24-48 hours using a dialysis bag with a molecular weight cutoff of 3-10 kDa to remove free small molecule byproducts. The dialysate is freeze-dried to obtain purified SA-His 22 product.

[0019] The pH-responsive mesenchymal stem cell exosome drug delivery system (pH-EVs) described in the present invention is structured as follows:

[0020] It consists of the following two parts: 1. Drug carrier exosomes (MSC-EVs), derived from human umbilical cord mesenchymal stem cells; 2. Functional modification molecule histidine peptide SA-His 22 , used to achieve pH-responsive release mechanism; The SA-His 22 Embedded in the phospholipid bilayer structure of MSC-EVs through hydrophobic interactions. Under acidic microenvironment conditions (such as tumor tissue or lysosomal environment), His 22Part of the protonation occurs, inducing structural relaxation, expansion or micropore formation in the EV membrane, thereby triggering the efficient release of the encapsulated drug and achieving environmentally sensitive controlled release.

[0021] The preparation method of the pH-EVs, a pH-responsive mesenchymal stem cell exosome drug delivery system according to the present invention, comprises the following steps: 1. Exosome isolation: Exosomes were extracted and purified from MSC cell culture supernatant using ultracentrifugation (120,000 × g, 2 h).

[0022] 2. Drug loading: Based on the physicochemical properties of PTX and Vacuolin-1, ultrasound, co-incubation, or other appropriate methods are selected to load the drug into MSC-EVs.

[0023] 3. Removal of free drugs: The drug-loaded EVs were subjected to ultracentrifugation again (120,000 × g, 2 h) to remove unbound free drugs.

[0024] 4.SA-His 22 Modification: The above EVs were mixed with pre-prepared SA-His 22 Mix and incubate at 37°C with gentle agitation for 1–2 h to promote stable insertion of SA into the EV membrane.

[0025] 5. Purification: After incubation, remove unbound SA-His by ultracentrifugation (120,000 × g, 2 hours). 22 molecules, and finally obtained functionally modified pH-responsive drug delivery systems (pH-EVs).

[0026] The pH-responsive mesenchymal stem cell exosome drug delivery system of the present invention can also have the following uses: 1. Application in tumor treatment pH-EVs can efficiently deliver a variety of chemotherapy drugs or immunotherapy molecules and trigger rapid release in the acidic microenvironment of tumors. The system combines the natural tumor chemotaxis of MSC-EVs and SA-His 22 Environmental responsiveness can effectively enhance drug concentration and reduce toxicity to normal tissues.

[0027] 2. Combination drug therapy The pH-EVs system of the application can not only precisely deliver PTX and Vacuolin-1 to the tumor site, but also inhibit the recruitment of MDSCs in the tumor microenvironment through the synergistic effect of PTX and Vacuolin-1, and improve the immunosuppression caused by chemotherapy. Vacuolin-1 enhances the anti-tumor response of the immune system by interfering with the metabolic interaction between tumor stromal cells and tumors. In addition, when PTX and Vacuolin-1 are used together, Vacuolin-1 can reduce the resistance of tumor stroma to therapeutic drugs and regulate the immune microenvironment, reduce immune escape caused by chemotherapy, and further improve the therapeutic effect. This combination therapy not only enhances the anti-tumor effect of paclitaxel (PTX), but also improves the tumor microenvironment by inhibiting the recruitment of immunosuppressive cells, providing an innovative precision treatment.

[0028] The application has the following significant technical advantages and application value: 1. Improve membrane anchoring stability and enhance surface functional modification efficiency By coupling long-chain fatty acid stearic acid (SA) to the His 22 rich N-terminal of the polypeptide, a functional module SA-His of hydrophobic-hydrophilic structure is constructed 22 . This structure can stably embed into the phospholipid bilayer membrane of exosomes and play the function of "lipid anchor", significantly enhancing the anchoring efficiency and stability of functional peptides on the membrane surface. Compared with unmodified His 22 , it has stronger membrane binding ability and significantly reduced shedding rate, thereby improving the consistency and delivery efficiency of the overall preparation.

[0029] 2. Enhance membrane penetration and cell uptake ability in acidic microenvironment SA-His 22 By protonation reaction of the histidine side chain in acidic environment, the local structure of exosome membrane is induced to be loose or microporous, enhancing the membrane permeability and drug release rate. At the same time, this reaction also promotes the fusion and endocytosis with the target cell membrane, improving the cell uptake efficiency and therapeutic activity of pH-EVs in acidic environments such as tumor tissues.

[0030] 3. Construct a composite delivery platform with high biocompatibility and high targeting SA-His 22 The composite system constructed by combining with MSC-EVs has good biocompatibility and low immunogenicity, and has the ability to actively chemotaxis to inflammatory and tumor sites. SA-His 22 further improves the functionalization level and surface activity of the exosome membrane, so that the system exhibits longer circulation time and better target recognition ability in vivo, which is significantly better than traditional artificial synthetic nanocarriers.

[0031] 4. Achieve multi-drug synergistic delivery and enhance the efficacy of therapeutic intervention The platform of this invention can simultaneously load different types of drugs, such as chemotherapeutics and autophagy inhibitors, to achieve multi-pathway synergistic therapy by jointly intervening in tumor cell proliferation, migration, and immune mechanisms. pH-EVs demonstrated excellent therapeutic responsiveness at various treatment time points, and in vivo experiments confirmed that they significantly reduced systemic toxicity while improving efficacy.

[0032] 5. Achieve precise temporal and spatial pH-responsive release control SA-His 22 The histidine structure of the drug is highly pH-sensitive, triggering protonation reactions in acidic conditions such as the tumor microenvironment or lysosomes, altering hydrophobicity and inducing EV membrane reorganization, thereby precisely regulating the timing and spatial distribution of drug release. This mechanism reduces the risk of nonspecific drug distribution in normal tissues while significantly increasing local drug concentration and therapeutic efficiency at the tumor site.

[0033] 6. Excellent safety and clinical translation potential The MSC-EVs used in the present invention are derived from human umbilical cord mesenchymal stem cells, have low immunogenicity and are non-tumorigenic, thus avoiding the possible interference of carcinogenic signals caused by EVs derived from tumor cells. 22 It is artificially synthesized, has a clear structure, and controllable reactions. The delivery system formed by its combination with EVs has shown excellent safety in multiple in vivo and in vitro experiments, providing a solid foundation for future clinical transformation.

[0034] The system of the present invention introduces a histidine peptide modified by stearic acid (SA-His 22 ), giving it pH-responsive release capability in a specific acidic microenvironment. 22 The histidine residues in the structure are protonated under acidic conditions, triggering the microstructural reorganization of the EV membrane (such as membrane micropores or swelling), thereby achieving controlled release of drugs at the targeted lesion site and improving the accuracy and efficiency of treatment.

[0035] More importantly, this membrane modification strategy also significantly enhanced the uptake of EVs in target cells, especially under acidic conditions such as tumor microenvironment, where the charge change and structural adaptation transformation of the membrane surface can promote the fusion and internalization of exosomes with cell membranes. Flow cytometry and confocal microscopy imaging results showed that the SA-His 22 The functionally modified exosomes showed higher cellular uptake rates in Panc02 and PSC cells, especially PSC cells, which have stronger vesicle internalization ability in the tumor microenvironment, giving the system of the present invention a stronger matrix-targeted delivery advantage.

[0036] The present invention is achieved by 22 The hydrophobic stearic acid (SA) molecule was introduced into the N-terminus to construct the amphiphilic structural unit SA-His 22 This structure can be stably inserted into the EV phospholipid bilayer through hydrophobic interaction, significantly improving the peptide anchoring efficiency and enhancing its membrane modification stability. At the same time, this modification strategy also gives EVs membrane responsiveness, allowing it to undergo structural reconstruction in acidic microenvironments (such as tumor TME or intracellular lysosomes), thereby achieving precise release of the drug delivery system. More importantly, SA-His 22 The modification also significantly enhanced the cellular uptake of EVs in acidic environments. After protonation on the membrane surface, their surface charge and structural state changed, promoting fusion with the target cell membrane and internalization.

[0037] The positive effect of the present invention is that: the SA-His 22 The modified pH-responsive mesenchymal stem cell exosome drug delivery system (pH-EVs) not only solves the technical difficulties of existing delivery platforms such as imprecise drug release, low release efficiency and unstable membrane modification, but also enhances the cellular uptake efficiency of exosomes through membrane potential regulation and structural sensitivity. It also improves the effect of myeloid-derived suppressor cell recruitment at the tumor site on the immune response during chemotherapy through drug synergy. It is particularly suitable for multi-target delivery and combination therapy in the complex microenvironment of solid tumors such as pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 :Western blot was used to detect marker proteins of exosomes to verify the purity of exosomes; Figure 2 :Mass spectrometry detection of SA-His 22 Whether the stearic acid modification is successful; Figure 3 :HPLC detection of His before and after modification 22 purity; Figure 4 : Exosome particle size before and after modification; Figure 6 : Changes in particle size and potential of pH-EVs under different acidic environments; Figure 5 :Transmission electron microscopy was used to observe the morphology of MSC-EVs and pH-EVs before and after modification; Figure 7 : Comparison of drug release rates of pH-EVs under neutral and acidic environments; Figure 8 :Uptake of MSC-EVs and pH-EVs by tumor cells and stellate cells under neutral and acidic environments; Figure 9:Comparison of the in vivo and tissue distribution of pH-EVs and MSC-EVs in mice; Figure 10 : Immunofluorescence sections of pH-EVs in tumor sites; Figure 11 :Confocal microscopy was used to observe the expression of Casapase-3 and LC3B in the co-culture model of tumor cells and stellate cells; Figure 12 :The ability of Vacuolin-1 to inhibit the binding of LC3 to lysosomes in astrocytes was observed by confocal microscopy; Figure 13 : The killing ability of pH-EVs on tumor cells; Figure 14 :The anti-proliferative ability of pH-EVs in a co-culture model of tumor cells and stellate cells; Figure 15 : Anti-migration ability of pH-EVs in a co-culture model of tumor cells and stellate cells; Figure 16 :WB expression changes of Ccl2 in tumor sites after mouse treatment; Figure 17 :Flow cytometry was used to detect the recruitment ratio of myeloid-derived suppressor cells in the tumor site after treatment in mice; Figure 18 :Immunofluorescence sections were used to detect the changes of Ccl2 and myeloid-derived suppressor cells in the tumor sites of mice after treatment; Figure 19 :In vivo imaging detection of mouse tumor fluorescence intensity during drug administration; Figure 20 : Ex vivo images of tumor volume; Figure 21 : The body weight of mice was measured during the drug administration process. DETAILED DESCRIPTION

[0039] The present invention is further described by way of examples below, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or alteration of the present invention that can be easily implemented by a person skilled in the art will fall within the scope of the claims of the present invention.

[0040] Example 1: SA-His 22 Synthesis, purification and structure identification of 1) Exosome isolation: Exosomes were extracted and purified from MSC cell culture supernatant using ultracentrifugation (120,000 × g, 2 h); 2) Drug loading: Based on the physicochemical properties of the drug to be delivered, ultrasound, co-incubation, or other appropriate methods are used to load the drug into MSC-EVs. 3) Removal of free drugs: The drug-loaded EVs were subjected to ultracentrifugation again (120,000 × g, 2 h) to remove unbound free drugs; 4) SA-His 22 Modification: The above EVs were mixed with pre-prepared SA-His 22 Mix and incubate at 37°C with gentle agitation for 1–2 h to promote the stable insertion of SA into the EV membrane structure; 5) Purification: After incubation, unbound SA-His was removed by ultracentrifugation (120,000 × g, 2 hours). 22 molecules, and finally obtained functionally modified pH-responsive drug delivery systems (pH-EVs).

[0041] Example 2: Preparation and characterization of pH-EVs

[0042] To obtain pure exosome-depleted medium, fetal bovine serum was first ultracentrifuged at 100,000 × g for 3 hours to remove endogenous exosomes (EVs). Mesenchymal stem cells (MSCs) were cultured with the treated medium for 48 hours, and the supernatant was collected and centrifuged at 2,000 × g for 10 minutes and then at 10,000 × g for 20 minutes to remove cellular debris. The supernatant was then filtered through a 0.22 µm filter and concentrated by ultrafiltration. Finally, the pellet was centrifuged at 100,000 × g for 2 hours to collect EVs.

[0043] The obtained EVs were resuspended in PBS and their marker proteins (such as CD63, Alix, TSG101) and negative proteins (such as Calnexin) were detected by Western blot. The results showed that ( Figure 1 The EVs are of high purity. The MSC-EVs produced are derived from human umbilical cord mesenchymal stem cells and are low in immunogenicity and non-tumorigenic, thus avoiding the potential oncogenic signaling interference from EVs derived from tumor cells.

[0044] Before surface functionalization of EVs, stearic acid active ester (SA-NHS) was first synthesized. Stearic acid and N-hydroxysuccinimide (NHS) were dissolved in anhydrous dimethylformamide (DMF) at a molar ratio of 1:1.5 and stirred at room temperature for 4–8 hours. The resulting reaction solution was used directly for the coupling reaction or the solvent was removed by rotary evaporation to obtain the purified SA-NHS intermediate. Subsequently, SA-NHS was slowly added dropwise to the His-containing 22 After the reaction is completed, the peptide is dialyzed in deionized water for 24–48 hours using a dialysis bag with a molecular weight cutoff of 3–10 kDa to remove free impurities. The dialyzed product is finally lyophilized to obtain SA-His.22 Solid product. The molecular weight change was detected by time-of-flight mass spectrometry (MALDI-TOF). Figure 2 ), and its high purity and structural correctness were confirmed by high performance liquid chromatography (HPLC) ( Figure 3 ), verifying that SA was successfully coupled to His 22 , which is suitable for further modification and application. By coupling the long-chain fatty acid stearic acid (SA) to the histidine-rich His 22 The functional module SA-His at the N-terminus of the polypeptide builds a hydrophobic-hydrophilic structure 22 This structure can be stably embedded in the exosome phospholipid bilayer membrane, play the role of "lipid anchor", and significantly enhance the anchoring efficiency and stability of functional peptides on the membrane surface. The above-mentioned substance (specifically named stearic acid-histidine peptide SA-His 22 ) has the following molecular structure: .

[0045] Subsequently, paclitaxel (PTX) and vacuolin-1 were loaded into EVs, and drug encapsulation was achieved by sonication (10s×10 times) and incubation at 37°C for 1 hour. Free drugs were then removed by centrifugation at 12,000 rpm. SA-His22 was then added for a second incubation, followed by centrifugation at 20,000×g for 2 hours to remove unbound SA-His22 molecules and obtain purified pH-responsive EVs (pH-EVs). Nanoflow cytometry and Malvern Zeta particle size analysis were used to characterize the particle size of EVs before and after modification. Figure 4 ), the results showed that SA-His22 modification did not affect the structural stability of EVs. Under pH 6.5 conditions, the particle size of pH-EVs increased and the potential tended to be positive, indicating that it has structural responsiveness in an acidic environment ( Figure 5 EVs before and after modification were imaged using transmission electron microscopy ( Figure 6 ), the results showed that SA-His22 modification did not destroy the vesicle morphology and membrane structure of EVs, maintaining their integrity and natural characteristics.

[0046] SA-His 22 The composite system (pH-EVs) constructed by combining with MSC-EVs has good biocompatibility and low immunogenicity. 22 The functionalization level and surface activity of the exosome membrane have been further improved, so that the system will exhibit a longer circulation time and better target recognition ability in the body, which is significantly better than traditional artificially synthesized nanocarriers.

[0047] The positive effects of the present invention are demonstrated by the following experimental examples: Experimental Example 1: High uptake performance of pH-EVs and tumor microenvironment response mechanism

[0048] To evaluate the drug release performance of the constructed pH-responsive exosomes (pH-EVs), paclitaxel (PTX)-loaded EVs and pH-EVs were placed in dialysis bags with a molecular weight cutoff of 10 kDa, suspended in PBS buffer at pH 6.5, and subjected to in vitro sustained release experiments at 37°C and 100 rpm. Samples were taken at multiple time points, and the drug concentration in the released solution was determined by high-performance liquid chromatography (HPLC). Cumulative release curves were plotted ( Figure 7 The results showed that pH-EVs exhibited significantly faster and more complete drug release in an acidic environment. Compared with ordinary EVs, their release rate and cumulative release amount were significantly improved, verifying that SA-His22 modification gave them good pH-responsive properties.

[0049] To further explore the targeting properties of pH-EVs in cellular uptake, flow cytometry and confocal microscopy were used to evaluate their uptake by pancreatic cancer cells (Panc02) and pancreatic stellate cells (PSC) under different pH conditions. Fluorescently labeled pH-EVs and MSC-derived EVs were incubated in Panc02 and PSC cells at pH 7.4 and pH 6.5, respectively, to measure the cellular uptake efficiency ( Figure 8 The experimental results showed that in the acidic microenvironment of pH 6.5, the uptake rate of EVs was significantly improved, especially the uptake level of pH-EVs in PSC cells was significantly better than that in Panc02 cells, suggesting that the system has stronger cell targeting ability in the tumor microenvironment (especially stromal cells). 22 The protonation reaction of the histidine side chain in an acidic environment induces local loosening of the exosome membrane structure or formation of micropores, enhancing membrane permeability and drug release rate. At the same time, this reaction also promotes the fusion and internalization of the exosome membrane with the target cell membrane, improving the cellular uptake efficiency and therapeutic activity of pH-EVs in acidic environments such as tumor tissue.

[0050] In the in vivo distribution study, pH-EVs and MSC-EVs loaded with fluorescently labeled PTX and Vacuolin-1 were administered to pancreatic cancer-bearing mice via tail vein injection. The distribution of EVs in vivo was tracked using a small animal fluorescence imaging system 2 hours and 4 hours after administration, respectively. Figure 9The results showed that pH-EVs had more obvious fluorescent signal enrichment in the tumor area and exhibited better tumor active localization and retention capabilities compared to other organs and tissues, indicating that it has good in vivo targeted delivery properties.

[0051] To verify the drug delivery effect of pH-EVs in tumor tissue, tumor tissues were fixed, paraffin-embedded, serially sectioned, and immunofluorescence stained. Confocal microscopy was performed after using DAPI to stain the nucleus and quenching agent to seal the sections ( Figure 10 The results showed that pH-EVs can be effectively distributed in tumor tissues and successfully release the loaded drugs, showing good intratumoral delivery and tissue penetration capabilities, further confirming its responsive release mechanism in the slightly acidic tumor microenvironment.

[0052] Conclusion: In summary, pH-EVs have excellent pH-triggered drug release ability in vitro, and show good tumor-targeted uptake and delivery efficiency at the cellular level and in animal models, especially with a stronger uptake tendency for tumor stromal cells, showing its potential application value in precision anti-tumor therapy.

[0053] Experimental Example 2: Verification of the Anti-tumor Effect and Analysis of Therapeutic Potential of pH-EVs

[0054] To evaluate the drug effects of paclitaxel (PTX) and Vacuolin-1, the present invention constructed a Panc02-PSC co-culture system and evaluated the expression of apoptosis protein Caspase-3 and autophagy protein LC3B in different treatment groups using confocal microscopy. The results showed that the combined treatment of PTX and Vacuolin-1 significantly enhanced the activity of Caspase-3 and significantly reduced the expression of LC3B in the Panc02-PSC co-culture system, indicating that cell death was enhanced and autophagy inhibition was significantly improved ( Figure 11 ). On this basis, the present invention further explored the mechanism of action of Vacuolin-1. Low-glucose autophagy induction experiments were conducted on drug-treated PSC cells, and Vacuolin-1 was added for combined treatment. After treatment, lysosome staining and LC3B staining were used for observation. The results showed that Vacuolin-1 significantly inhibited the fusion of lysosomes and autophagosomes, interfered with the function of lysosomes, and thus effectively inhibited the autophagy process of cells ( Figure 12 This finding further supports the ability of Vacuolin-1 to inhibit autophagy in tumor stromal cells and its ability to interfere with lysosomal function, providing new ideas for tumor treatment.

[0055] On the basis of the above research, in order to systematically evaluate the anti-tumor activity of pH-responsive exosomes (pH-EVs), their anti-cell proliferation effect was first verified in an in vitro model. Pancreatic cancer cells (Panc02) and pancreatic stellate cells (PSC) were seeded in 96-well plates, and after the cells were stably attached to the wall, different treatment groups were added: EVs loaded with paclitaxel only (EVs-PTX), EVs loaded with paclitaxel and Vacuolin-1 (EVs-P / V), and pH-EVs modified with SA-His22. After 24 hours, 48 ​​hours and 72 hours, the cell killing ability of each treatment group was detected by MTT assay ( Figure 13 ). The results showed that the three drug treatment groups could inhibit cell viability to varying degrees, among which the pH-EVs group showed significantly higher cytotoxicity at all time points, especially in PSC cells. The inhibitory effect was most prominent. Next, the scratch healing area and bioluminescence measurements in Panc02 and Panc02-Luc cells were used to evaluate the anti-migratory and anti-proliferative effects of pH-EV. The results showed that PSC promoted the migration and proliferation of Panc02 under control conditions. Among the treatment groups, the combined action group of PTX and Vacuolin-1 had obvious anti-migratory and anti-proliferative abilities, among which pH-EV showed the most significant therapeutic effect ( Figures 14-15 This suggests that pH-EVs not only have good drug release properties, but also enhance the anti-tumor stromal cell effect due to their environmental responsiveness and targeting ability, helping to destroy the tumor microenvironment and thus improving the overall anti-tumor efficiency.

[0056] The present invention further verified the inhibitory effect of the pH-EVs system on myeloid-derived suppressor cells (MDSCs) in a pancreatic cancer tumor-bearing mouse model. The successfully constructed mouse model was randomly divided into groups and given the following treatments: exosomes loaded with paclitaxel only (EVs-PTX), exosomes loaded with paclitaxel and Vacuolin-1 (EVs-P / V), pH-responsive exosomes (pH-EVs) and a blank control group. All mice were regularly dosed via tail vein injection. After drug administration, the present invention performed Western blotting analysis on the tumor tissue, and the results showed that Ccl2 was significantly increased in the EVs-PTX treatment group, while Ccl2 protein expression was significantly decreased in the EVs-P / V and pH-EVs treatment groups, suggesting that PTX would increase Ccl2 expression, and this result was improved after the introduction of Vacuolin-1 combined treatment, and this inhibitory effect was significantly amplified after the addition of the excellent delivery system of pH-EVs ( Figure 16Subsequently, flow cytometry was used to analyze the cell populations within the tumor tissue. The results showed that in the EVs-PTX treatment group, the proportion of MDSCs in the tumor site was significantly increased. This phenomenon was significantly improved in the EVs-P / V combined treatment group with the introduction of Vacuolin-1. After further introduction of the pH-EVs drug delivery system, the proportion of MDSCs in the tumor site decreased again ( Figure 17 Further immunofluorescence analysis showed that the expression of CCL2 and the infiltration of MDSCs were significantly reduced in the EVs-P / V combined treatment group, while the infiltration of CD8+ T cells increased. This effect was particularly significant in the pH-EVs group ( Figure 18 The results are mutually verified with the above experimental results, further demonstrating the strong potential of pH-EVs in inhibiting MDSCs recruitment and promoting anti-tumor immune response.

[0057] The in vivo anti-tumor effect was further verified in a pancreatic cancer-bearing mouse model. The mice with successful modeling were randomly divided into groups and treated with EVs-PTX, EVs-P / V, pH-EVs, and a blank control group, respectively. The drugs were administered regularly via tail vein injection. The weight of the mice was continuously monitored during the experiment to assess the systemic toxicity of the drug, and tumor bioluminescence imaging was performed regularly during the administration. Figure 19 ) to dynamically monitor tumor growth. At the end of the experiment, tumor tissue was dissected, weighed, and statistically analyzed ( Figure 20 –21). The results showed that the pH-EVs group was most effective in inhibiting tumor volume growth, with a significantly reduced fluorescence signal in tumor tissue and the lowest endpoint tumor weight. No significant weight loss or toxicity-related symptoms were observed throughout the treatment cycle, indicating that this delivery system has good biosafety while enhancing anti-tumor effects.

[0058] Conclusion: Based on in vitro and in vivo experimental results, the pH-EVs system, through its unique carrier design and the pH-responsive nature of SA-His22 modification, enables precise drug release within the acidic tumor microenvironment. The targeted delivery advantages of this system effectively enhance the synergistic effect of paclitaxel (PTX) and Vacuolin-1, increasing drug efficacy while minimizing the effects on normal tissues. In particular, Vacuolin-1 enhances the anti-tumor effect of PTX by interfering with lysosomal function, inhibiting autophagy and Ccl2 expression, further improving the tumor microenvironment. Therefore, pH-EVs not only optimize drug delivery efficiency but also, through drug synergy, provide a novel strategy for the precision treatment of solid tumors such as pancreatic cancer. Therefore, the application prospects of pH-EVs in the treatment of solid tumors such as pancreatic cancer warrant further in-depth research and clinical translation.

[0059] Paclitaxel (PTX) chemotherapy has demonstrated remarkable therapeutic efficacy in a variety of solid tumors, but increasing evidence suggests that this treatment inadvertently contributes to the establishment of an immunosuppressive tumor microenvironment. A key mechanism underlying this is paclitaxel-induced expansion and recruitment of myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature myeloid cells known for their ability to suppress T cell-mediated anti-tumor immune responses. Recent studies have demonstrated that paclitaxel and other chemotherapeutic agents can trigger systemic inflammation and stromal remodeling, thereby promoting the mobilization of MDSCs from the bone marrow to peripheral tissues, including tumor sites. This recruitment not only impairs the efficacy of cytotoxic therapies but also interferes with emerging immunotherapy strategies, presenting a major obstacle to achieving durable clinical responses. Therefore, elucidating the molecular mechanisms by which paclitaxel drives MDSC mobilization and function is crucial for developing combination strategies that can mitigate its pro-tumor side effects and enhance overall therapeutic efficacy. This study incorporates the small molecule Vacuolin-1 into paclitaxel-based pancreatic cancer treatments, aiming to simultaneously inhibit autophagy mediated by tumor stromal cells (PSCs), disrupt stromal-tumor metabolic interactions, and mitigate chemotherapy-induced MDSC-driven immunosuppression. Vacuolin-1 is a highly selective autophagy inhibitor that significantly inhibits autophagic flux by blocking autophagosome-lysosome fusion and exhibits potent activity in various cell models. Unlike traditional autophagy inhibitors that primarily target upstream regulatory factors, Vacuolin-1 specifically targets the late stages of the autophagic process. It stabilizes the lysosomal membrane structure and prevents autophagosome-lysosome fusion, thereby minimizing nonspecific interference with basal metabolic functions. Given that the mechanism by which paclitaxel induces MDSC recruitment involves Ccl2 expression, Vacuolin-1 may not only act by inhibiting autophagosome-lysosome fusion but also by interfering with lysosomal function, inhibiting Ccl2 production and thereby reducing MDSC recruitment. Through this mechanism, Vacuolin-1 may reshape the immune microenvironment while reducing tumor stromal cell-mediated autophagy support, further reducing the immunosuppressive effects caused by chemotherapy.

[0060] Although the present invention has been described in detail through specific embodiments, these embodiments are merely preferred or exemplary embodiments of the present invention and do not limit the scope of protection of the present invention. In actual applications, those skilled in the art may make equivalent modifications and optimizations to various details of the present invention without departing from the technical ideas and innovations of the present invention.

Claims

1. A histidine peptide SA-His 22 , which is characterized by Has the following structure: 。 2. A histidine polypeptide SA-His according to claim 1 22 The preparation method comprises the following steps: 1) SA activation reaction: Dissolve octadecanoic acid and an equimolar amount of N-hydroxysuccinimide in anhydrous dimethylformamide and stir at room temperature for 4–8 hours to generate the active ester SA-NHS; 2) His 22 Coupling reaction: His 22 Dissolve in a small amount of buffer, slowly add SA-NHS solution, and continue to react at room temperature or under appropriate conditions for several hours to achieve directional coupling of the N-terminal amino group; 3) Purification: After the reaction, dialyze the product in deionized water for 24–48 hours using a dialysis bag with a molecular weight cutoff of 3–10 kDa to remove free small molecule byproducts. The dialyzed fluid was lyophilized to obtain the purified SA-His22 product.

3. A pH-responsive mesenchymal stem cell exosome drug delivery system, characterized in that It mainly consists of the following two parts composition: 1) Drug carrier exosomes (MSC-EVs): derived from human umbilical cord mesenchymal stem cells; 2) Functional modified molecule histidine peptide SA-His as claimed in claim 1 22 : Used to achieve pH-responsive release mechanism; The histidine polypeptide SA-His 22 Embedded in the phospholipid bilayer structure of MSC-EVs through hydrophobic interactions; Under acidic microenvironment, His 22 Part of the protonation occurs, inducing structural relaxation, expansion or micropore formation in the EV membrane, thereby triggering the efficient release of the encapsulated drug and achieving environmentally sensitive controlled release.

4. The method for preparing a pH-responsive mesenchymal stem cell exosome drug delivery system according to claim 1, comprising the following steps: 1) Exosome isolation: Exosomes were extracted and purified from MSC cell culture supernatant using ultracentrifugation (120,000 × g, 2 h); 2) Drug loading: Based on the physicochemical properties of the drug to be delivered, ultrasound, co-incubation, or other appropriate methods are used to load the drug into MSC-EVs. 3) Removal of free drugs: The drug-loaded EVs were subjected to ultracentrifugation again (120,000 × g, 2 h) to remove unbound free drugs; 4) SA-His 22 Modification: The above EVs were mixed with pre-prepared SA-His 22 Mix and incubate at 37°C with gentle agitation for 1–2 h to promote the stable insertion of SA into the EV membrane structure; 5) Purification: After incubation, unbound SA-His was removed by ultracentrifugation (120,000 × g, 2 hours). 22 molecules, and finally obtained functionally modified pH-responsive drug delivery systems (pH-EVs).

5. Use of the pH-responsive mesenchymal stem cell exosome drug delivery system according to claim 1 in the preparation of a drug for treating tumors.

6. Use of the pH-responsive mesenchymal stem cell exosome drug delivery system according to claim 1 in the preparation of a drug for treating pancreatic cancer.

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