Abstract Background Extracellular vesicles (EVs) and extruded nanovesicles (ENVs) are promising nanovesicles (NVs) for drug delivery. However, the application of these NVs is strongly hindered by their short half-life in the circulation. Macrophages (Mφs) in the liver and spleen contribute to the rapid depletion of NVs, but the underlying mechanism is unclear. Methods By collecting the supernatant of PANC-1 cells and squeezing PANC-1 cells, EVs and ENVs derived from PANC-1 cells were prepared via ultracentrifugation. NVs were subsequently identified via western blot, particle size measurement, and electron microscopy. The distribution of NVs in mouse bodies was observed with a live animal imaging system. Liver Mφs were extracted and isolated after NVs were administered, and transcriptome profiling was applied to determine differentially expressed genes (DEGs). siRNAs targeting interested genes were designed and synthesized. In vitro experiments, Mφs were transfected with siRNA or treated with the corresponding inhibitor, after which NV uptake was recorded. Doxorubicin (DOX) was encapsulated in ENVs using an ultrasound method. PANC-1 cell-derived tumors were established in nude mice in vivo, inhibitor pretreatment or no treatment was administered before intravenous injection of ENVs-DOX, and the therapeutic efficacy of ENVs-DOX was evaluated. Results NVs derived from PANC-1 cells were first prepared and identified. After intravenous injection, most NVs were engulfed by Mφs in the liver and spleen. Seven genes of interest were selected via transcriptome sequencing and validated via RT‒PCR. These results confirmed that the TLR2 signaling pathway is responsible for phagocytosis. siTLR2 and its inhibitor sparstolonin B (SpB) significantly inhibited the internalization of NVs by Mφs and downregulated the activity of the TLR2 pathway. The accumulation of ENVs-DOX in the liver was inhibited in vivo by pretreatment with SpB 40 min before intravenous injection, ultimately delaying tumor progression. Conclusion The TLR2 pathway plays a crucial role in the sequestration of NVs by Mφs. A novel antiphagocytic strategy in which pretreatment of mice with SpB inhibits the clearance of NVs and prolongs their half-life in vivo, thereby improving delivery efficiency, was identified. Graphical Abstract [46]graphic file with name 12951_2024_3001_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12951-024-03001-6. Keywords: Nanovesicles, Macrophages, Phagocytosis, Toll-like receptor 2, Drug delivery Backgrounds Extracellular vesicles (EVs) are a type of lipid bilayer nanoparticle derived from the cell membrane [[47]1–[48]3]. In general, EVs include naturally generated vesicles, such as exosomes, large vesicles, and apoptotic bodies. Owing to their unique structural characteristics, powerful drug loading ability, predominant physiochemical stability and biocompatibility properties, EVs have become promising tools in nanomedicine [[49]1, [50]2, [51]4, [52]5]. Among them, Exosomes have been extensively studied for therapeutic and diagnostic delivery [[53]4, [54]5]. In order to overcome the demerit of low output of exosomes, researchers have produced artificial vesicles by mechanically extruding donor cells to produce many microbubbles with increased efficiency. Accumulating evidence has shown that extruded nanovesicles (ENVs) are good substitutes for EVs [[55]6–[56]8]. In this study, both EVs and extruded nanovesicles (ENVs) were termed nanovesicles (NVs). The original purpose of developing nanocarriers was to reduce the nonspecific accumulation of agents and improve treatment efficacy. Compared with free medicinal substances, NVs can alter the distribution of drugs in the body and reduce systemic toxicity; however, the delivery efficiency of NVs is far from satisfactory. There is a consensus that most administered NVs are rapidly cleared by the mononuclear phagocyte system (MPS), mainly by macrophages (Mφs), and that only a very small number of injected nanoparticles reach the target tissues [[57]9–[58]11]. Takafumi Imai et al. reported that the depletion of Mφs in mice delays the clearance of intravenously administered B16BL6 exosomes from the blood [[59]10]. In nature, target cells and macrophages directly compete for NV uptake. Within a unit of time, the phagocytosis of nanoparticles by macrophages is reduced, leading to more opportunities for target cells to recognize and uptake NVs, and vice versa. Thus, the following topics should urgently be investigated: the mechanism underlying phagocytosis and methods to evade phagocytosis. The recognition and internalization of NVs by Mφs are related to interactions between the biological molecules on the surface of NVs and the receptors on Mφs [[60]12]. Modifying the membrane of NVs is a popular method used to alter their recognition and uptake [[61]13]. Proteins expressed on Mφs, such as CD36 (scavenger receptor B) [[62]14], CD169 [[63]15], and insulin-like growth factor receptor-1 [[64]16], and other molecules displayed on the surface of NVs, such as ɑνβ3/β5 integrins [[65]16], lectin galectin-5 and surface glycans [[66]16, [67]17], are involved in phagocytosis. The presence of exposed phosphatidylserine on the surface of NVs is an important “eat me” signal for Mφs [[68]18]. Annexin V, a classical phosphatidylserine-binding protein, is used to mask the phosphatidylserine of EVs and delay their clearance by macrophages [[69]19]. In addition, CD47, a fundamental “don’t eat me” signal, can interact with signal regulatory protein-α (SIRP-α) on Mφs and inhibit their activation [[70]20]. CD47 has been applied to decorate NVs in several studies, and enhanced retention of exosomes in the circulation has been observed [[71]21, [72]22]. Other potential candidates that exert anti-phagocytic effects include but are not limited to the following molecules: CD24, CD44, CD31, β2-microglobulin (β2 M), programmed cell death protein 1 (PD-1), antiphagocytic protein 1 (app1), and dehydroxymethylepoxyquinomicin (DHMEQ) [[73]23]. However, the methods used to modify NVs are usually inconvenient and inefficient. In this research, we explored the molecular mechanisms underlying the phagocytosis of NVs from the perspective of Mφs and attempted to develop a new strategy to prevent unexpected devouring by Mφs. In this study, NVs derived from PANC-1 cells were prepared and administered to mice. Then, liver Mφs derived from these mice were isolated and subjected to transcriptome analyses. The toll-like receptor 2 (TLR2) signaling pathway plays an important role, and siTLR2 and its inhibitor Sparstolonin B (SpB) significantly inhibits the uptake of NVs by Mφs. Materials and methods Cell line and culture conditions PANC-1 cells, a human pancreatic cancer cell line, were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in DMEM (Servicebio, China) supplemented with 10% fetal bovine serum (FBS; Bioexplorer, USA) and 1% penicillin‒streptomycin (Solarbio, China) in an incubator at 37 °C with 5% CO[2]. Isolation of EVs and ENVs derived from PANC-1 cells EV isolation PANC-1 cells were cultured to approximately 80% confluence, and the medium was replaced with DMEM supplemented with FBS without exosomes for another 48 h. The culture media was subsequently harvested, and the EVs in the medium were isolated via differential centrifugation as previously described [[74]24]. Preparation of ENVs PANC-1 cells were harvested, suspended in PBS at a density of 1 × 10^7 cells/ml and disrupted with an ultrasonic grinder (40% amplitude) for 120 cycles of 10 s on/5 s off. Next, the cell debris was serially squeezed with a liposome extruder (Avanti Polar Lipids, USA) through 10 μm, 5 μm and 1 μm polycarbonate membranes (Whatman, USA). Afterward, the suspension was centrifuged at 16,000 × g at 4 °C for 30 min to remove cell debris and filtered through a 0.22 μm pore filter. The obtained supernatant was ultracentrifuged at 100,000 × g at 4 °C for 60 min two times. The final pellet was ENVs. The amounts of EVs and ENVs were determined with a BCA assay, and the EVs and ENVs were stored at − 80 °C until use. Characterization of the EVs and ENVs The morphology of the NVs was imaged with a transmission electron microscope (TEM, Hitachi, Japan). A 20 µl EVs or ENVs suspension (1 µg/µl) was pipetted onto copper mesh and stained with 1% osmium tetroxide, then loaded the examples and photographed [[75]24]. The particle size distribution of these vesicles was analyzed on a Zetasizer Nano ZSP (Malvern, UK) and based on light intensity. The marker proteins in the EVs and ENVs were detected via western blot assay. Western blot analysis Total protein was extracted from the vesicles or cells with RIPA buffer containing a protease inhibitor mixture (Beyotime, China), quantified via a BCA assay (Biosharp, China), mixed with loading buffer (Beyotime, China) and stored at − 80 °C. The samples were subjected to western blot according to standard protocols. Briefly, protein samples were subjected to 5%~10% SDS‒PAGE and then transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were subsequently incubated with primary antibodies overnight as well as with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. Finally, the protein bands were visualized via a gel imaging system (Bio-Rad, USA). GAPDH or β-actin served as the internal control. The following antibodies were used: anti-Alix (Sigma, 1:10000), anti-CD81 (Univ-bio, 1:1000), anti-Tsg101 (Univ-bio, 1:1000), anti-TLR2 (Huabio, 1:1000), anti-MyD88 (Abcam, 1:1000), anti-NF-кB (Abcam, 1:1000), anti-pNF-кB (Abcam, 1:1000), anti-MAPK (Abcam, 1:1000), anti-pMAPK (Abcam, 1:1000), anti-GAPDH (Huabio, 1:5000) and anti-β-actin (Bioswamp, 1:5000). Live imaging assay All the animal studies were performed with the approval of the Ethics Committee of the Animal Experiment Center of Wuhan University. 6 ~ 8-week-old male athymic nude mice were purchased from Hunan SJA (Changsha, China) and were reared in a special pathogen-free (SPF) animal environment. To detect the pharmacokinetics of the NVs, the EVs or ENVs were labeled with DiR or DiI (Umibio, China). Specifically, the EVs or ENVs were coincubated with DiR or DiI at 37 °C for 20 min, and then ultracentrifuged at 100,000 × g at 4 °C for 60 min to discard the free DiR or DiI. The free dye or labeled NVs were injected into mice through the tail vein. Then the body distributions of the free dye or labeled NVs were assessed with a NightOWL II imaging system (Berthold, Germany). At given time points, the mice were anesthetized by inhaling a mixture of oxygen and isoflurane, then placed into the imaging machine and photographed. The fluorescence intensity was measured via IndiGO™ software. Immunofluorescence analysis Frozen sections of mouse tissues were prepared according to standard protocols. The tissue slides were blocked with 5% bovine serum albumin at room temperature for 1 h, and then incubated with an anti-mouse F4/80 antibody (Hua-bio, 1:200) and a CD68 antibody (Univ-bio, 1:200) overnight at 4 °C. After the slides were washed 3 times, they were stained with the corresponding secondary antibodies (Hua-bio, 1:200, FITC) for 1 h and with DAPI for 90 s. The slides were viewed via fluorescence microscopy. The fluorescence intensity and colocalization coefficients were analyzed via ImageJ software. Isolation of liver Mφs Mφs were isolated from the liver according to a standard protocol [[76]25]. Male 6 ~ 8-week-old BALB/c nude mice were euthanized by cervical dislocation, and the livers were immediately perfused with prewarmed DMEM (approximately 37 °C) containing 100 U/l collagenase IV (BioFroxx, Germany) via the inferior vena cava. The portal veins were cut open simultaneously. After thorough perfusion, the liver was removed and ground through 70 μm mesh to obtain a single-cell suspension. The hepatocytes were discarded by centrifugation at 50 × g at 4 °C for 5 min three times. The remaining precipitate containing the macrophages was subsequently resuspended in DMEM, and 3 ml of 70% Percoll (Solarbio, China), 3 ml of 30% Percoll, and 3 ml of the precipitated suspension were slowly and sequentially added. After centrifugation at 1800 × g at 4 °C for 20 min, the cell suspension was resuspended in DMEM supplemented with 10% FBS. After 0.5 h of incubation, the nonadherent cells were slowly washed away with PBS, and the remaining adherent cells were high-purity liver Mφs, which were cultured with specific medium (Procell, China). Transcriptome sequencing and data analysis Male 6 ~ 8-week-old BALB/c nude mice were treated with PBS, EVs or ENVs (n = 3 per group, intravenous administration). After 4 h, the liver Mφs were isolated via the method described above. Total RNA was isolated with TRIzol (Thermo, USA) according to the manufacturer’s instructions. The purity and concentration of the RNA were measured via a Nanodrop 2000 spectrophotometer (Nanodrop Technologies, USA). After passing the RNA sample test, subsequent RNA-Seq library preparation and sequencing were completed by Magigene Corporation (Guangzhou, China), and the 9 libraries were subjected to RNA sequencing on the Illumina HiSeq2500 high-throughput sequencing platform. Briefly, for the data analysis, the raw reads were aligned by discarding the low-quality sequences and ribosomal sequences. The obtained clean reads were further assembled and analyzed via StringTie. The DEGs were detected with DESeq, the default screening conditions were FDR ≤ 0.058 and |log2FC|≥1, and multiple testing correction was conducted via the Benjamini–Hochberg method. Finally, the DEGs were functionally annotated via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Quantitative real-time PCR (qRT–PCR) Total RNA derived from liver Mφs was purified, examined via the method described above and subsequently transcribed into cDNA with MonScript™ 5 × RT III All-in-One Mix (Monad, China). qRT‒PCR was conducted on a Bio-Rad Real-Time system using 2 × SYBR^® Green Pro Taq HS Premix (Agbio, China). The sequences of the primers used in this assay are listed in Table [77]S1. The GAPDHS was as internal reference. The relative expression values of other mRNAs were obtained via the 2^−∆∆Ct method. Internalization of NVs The NVs were stained with the PKH-67 (green fluorescent, Solarbio, China) according to the manufacturer’s instructions. Then, 30 µg of PKH-67-labeled ENVs was added to the medium of the liver Mφs (20,000 cells/well) or PANC-1 cells (20,000 cells/well). After 6 h, the cells were fixed with formalin solution (Servicebio, China), permeabilized with 0.5% Triton X-100 (Servicebio, China), stained with DAPI (nuclear dye; Servicebio, China), observed and imaged via fluorescence microscopy (LSM 780; Zeiss, Jena, Germany), and analyzed via Image J software (National Institute of Health, USA). siRNA transfection siRNAs against target genes were designed and synthesized by JTS Scientific (Beijing, China). The sequences of the three siRNAs used for each gene of interest are listed in Table [78]S2. RNA interference (RNAi) was conducted with the assistance of Hieff Trans^® siRNA/miRNA transfection reagent (Yeasen, China), and the synthesized siRNA or scramble control siRNA was transfected following recommended procedures to achieve the best transfection efficiency. Loading of DOX in ENVs and DOX release behavior For doxorubicin (DOX; Shanghai Yuanye, China) loading, different amounts of DOX were added to 1 ml ENVs (500 µg) solution, respectively. The mixture was disrupted via an ultrasonic grinder with 40% amplitude and 120 cycles of 10 s on/5 s off. Next, the mixture was kept at 37 °C for 1 h to restore the biofilm integrity of the ENVs. The ENVs were subsequently diluted with cold PBS and ultracentrifuged at 100,000 × g for 60 min at 4 °C to remove the unloaded free DOX. After the precipitate was resuspended in PBS, the encapsulated DOX concentration was determined by detecting the intrinsic fluorescence intensity of the liquid at 596 nm after excitation at 478 nm via an EnSight™ Multimode Microplate Reader (PerkinElmer, USA). The loading efficiency was calculated via the following equation: loading efficiency (%) = weight of loaded DOX in ENVs/weight of total DOX × 100%. For the DOX release study, 250 µl of ENVs-DOX was enclosed in a 14 kDa dialysis membrane bag (Bkmamlab, China) and incubated in 50 ml PBS (pH = 5.0 and 7.4, respectively) at 37 °C on a shaker. At regular intervals, the concentration of DOX in the PBS was measured via the absorbance method described above. Drug release was quantified via the following equation: drug release (%) = DOX amount in release media/DOX amount in ENV × 100%. Cytotoxicity assay A cell counting kit (CCK)-8 assay was used to investigate the cytotoxicity of ENVs-DOX and DOX to PANC-1 cells. PANC-1 cells were transplanted into 96-well plates (3000 cells/well). The next day, the cells were exposed to different concentrations of DOX or ENVs-DOX (containing the same amount of DOX) for 24 h. Next, the culture medium was discarded, CCK-8 reagent was added (Biosharp, China), the cells were incubated for an additional 2 h, and the optical density at 450 nm was subsequently measured via a microplate reader (Perkin Elmer, UK). Detecting ENVs in blood circulation To determine the residence time of ENVs in the peripheral blood, DiR-labeled ENVs (3 mg/kg) were intravenous injected into nude mice receiving oil/DMSO or SpB (24 mg/kg) treatment. Next, 80 µl of blood was collected into an anticoagulant EP tube through mouse orbit at different time points (0.5, 2, 4 and 6 h). The DiR signals were quantified via an EnSight™ Multimode Microplate Reader (excitation/emission = 748/780 nm). Animal experiments Male 3 ~ 5-week-old athymic nude mice were obtained from Hunan SJA (Changsha, China) and reared in an SPF animal environment. A total of 1 × 10^7 PANC-1 cells were collected in 300 µl of PBS and inoculated into the left flank of each mouse. The tumor masses were visible approximately 14 days after implantation, and the tumor-bearing mice were randomly divided into 6 groups (n = 5) to receive the following treatments: (1) oil/DMSO + PBS was used as a control; (2) oil/DMSO + free DOX; (3) SpB + PBS; (4) oil/DMSO + blank ENVs; (5) oil/DMSO + ENVs-DOX and (6) SpB + ENVs-DOX. SpB (MedChemExpress, USA) was dissolved in DMSO (Psatong, China) and diluted with corn oil (Psatong, China) before use. The mice were pretreated with oil/DMSO or SpB through intraperitoneal injection, and PBS, DOX or ENVs-DOX were subsequently injected through the tail vein 40 min later. The mice were injected with free DOX at a dose of 1 mg/kg body weight or with ENVs-DOX containing an equal dose of DOX. The treatment were conducted every 3 days for 2 weeks. The tumor size was examined every 2 days (volume = length × width^2/2). The mice were sacrificed, after which the subcutaneous xenografts, main organs and blood samples were harvested for histopathological and immunohistological staining. Hematoxylin and eosin (H&E) staining The histopathological analysis was performed following the standard process. The collected liver, kidneys, spleen and heart were fixed in 4% polyformaldehyde for 24 ~ 48 h, dehydrated in alcohol, embedded in paraffin, and cut into 4-µm-thick slides. Sections were stained with hematoxylin and eosin (H&E). The sections were diagnosed by experienced pathologists, and images were captured via an optical microscope (Olympus BX-40, Japan). Biochemistry analyses Plasma was obtained after centrifuging the whole blood at 3000 rpm for 15 min and stored at − 20 °C until use. Biochemical examinations were conducted via spectrophotometric analysis with a serum multiple biochemical analyzer (Siemens, Germany). Hepatic function was assessed through alanine aminotransferase (ALT) assay, and renal damage was determined with urea and creatinine (CRE) concentration measurements, whereas cardiac toxicity was evaluated through creatine kinase isoform MB (CK-MB) quantification [[79]26, [80]27]. Measurement of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) levels Liver Mφs were stimulated with EVs or ENVs, and the supernatant was collected after 6 h. IL-6 and TNF-α levels in the supernatants and LPS levels in the serum were determined with an enzyme-linked immunosorbent assay (ELISA) kit (Servicebio, China) following the manufacturer’s protocol. Statistical analysis All the experimental data were presented as the mean ± SD. Graphs and statistical analyses were generated with GraphPad Prism 7 software (GraphPad Software, USA). The significance of differences in the means between individual groups was analyzed by one-way or two-way analysis of variance. Differences between two groups were analyzed by the student’s t test. P values < 0.05 were considered to indicate statistical significance. (* or # p < 0.05; ** or # # p < 0.01; *** or ### p < 0.001). Results Morphological characterization and identification of NVs EVs and ENVs derived from PANC-1 cells were prepared according to the methods presented in Fig. [81]1A. Figure [82]1B shows representative transmission electron microscopy (TEM) images of NVs; most of the vesicles were double concave, disc-shaped and smaller than 200 nm. Western blot analysis verified that well-defined EVs biomarkers (Alix, CD81 and Tsg101) were enriched in NVs (EVs and ENVs) and PANC-1 cells, EVs lacked the expression of β-actin, a housekeeping protein that usually presents in cells, there was also a weak expression of β-actin in ENVs, which might be explained by the fact that little skeletal proteins derived from donor cells enter ENVs during the rupture-recombination process (Fig. [83]1C). As displayed in the results of dynamic light scattering analysis, the average diameters of the EVs and ENVs were approximately132.1 nm and 127.7 nm, respectively, and the peak sizes in the distribution diagram were approximately 122 nm (Fig. [84]1D). Although EVs and ENVs are similar in morphology and characteristics, according to our data, the production of ENVs is approximately 25 times greater than that of EVs with the same cell count (96.39 ± 4.60 vs. 3.72 ± 0.51 µg, *** p < 0.001, Fig. [85]1E). Fig. 1. [86]Fig. 1 [87]Open in a new tab Preparation and characterization of extracellular vesicles (EVs) and extruded nanovesicles (ENVs). (A) Flow diagrams of the differential ultracentrifugation procedures for the isolation of EVs (up) and ENVs (down). (B) Transmission electron microscopy images of EVs and ENVs (68000×). The scale bar is 200 nm. (C) western blot analysis of the classical EV biomarker proteins CD81, Alix and Tsg101; β-actin is a negative marker. (D) The size distributions of EVs and ENVs were determined via dynamic light scattering (DLS) analysis, respectively. (E) The yields of EVs and ENVs derived from 1 × 10^7 PANC-1 cells (*** p < 0.001) NVs were engulfed mainly by Mφs in the liver Next, free DiR and DiR-labeled NVs derived from PANC-1 cells were administered to the mice via intravenous injection, and these mice were imaged in vivo after 1 h. As shown in Fig. [88]2A & B, a remarkable increase in fluorescence signals was detected in the liver, spleen and lung. After free DiR was injected, the fluorescence signals in the lungs and spleen were greater than those after DiR-labeled NVs were injected, and the biodistribution of EVs and ENVs was not obviously different (Fig. [89]2C). To determine the specific cell population devouring the DiI-labeled NVs (EVs and ENVs), F4/80, a recognized surface marker of mononuclear phagocytes, was used to stain the macrophages. As shown in Fig. [90]2D, most red fluorescence derived from the DiI-labeled ENVs colocalized with the green signals from the F4/80^+ cells in the liver, spleen and lungs. The results for the DiI-labeled EVs were similar (Fig. [91]S1A& B). The colocalization analysis results of fluorescent pictures showed in Fig. [92]S1C. These results suggest that Mφs are the main cell type that engulfs EVs and ENVs in the circulation. Fig. 2. Fig. 2 [93]Open in a new tab Body distribution of EVs and ENVs. (A) DiR solution (1 µl stock solution was diluted to 100 µl), DiR-labeled EVs and DiR-labeled ENVs (100 µg/100 µl) were injected into nude mice via the caudal vein, respectively (n = 3 each group). After 1 h of intravenous administration, the mice were subjected to live imaging scanning with a NightOWL II Imaging System. The fluorescence signals indicate the body distribution of free DiR (left), DiR-labeled EVs (middle) and DiR-labeled ENVs (right). (B & C) 4 h after injection, the mice were sacrificed, and representative images of various organs, including the bowels, liver, lung, spleen, kidney, and heart, were obtained (C). The fluorescence intensity of each organ was measured via IndiGO™ software (*p < 0.05, **p < 0.01, *** p < 0.001). (D) The representative immunofluorescence images of ENVs group. DiI-labeled EVs and DiI-labeled ENVs (100 µg/100 µl) were injected into nude mice via the caudal vein, respectively (n = 3 each group). The mice were euthanized 4 h later, livers, spleens and lungs were harvested. Frozen liver, spleen and lung sections were stained with F4/80 antibodies. F4/80 (green, he excitation filter/emission filter = 475 ~ 490/505 ~ 535 nm) was used to indicate macrophages, and the red signals (he excitation filter/emission filter = 560 ~ 580/600 ~ 650 nm) indicate free DiI or DiI-labeled NVs. The scale bar represents 40 μm. The images of other groups could be found in Figure [94]S1 (*** p < 0.001) Whole-transcriptome sequencing revealed that the differentially expressed genes (DEGs) were enriched Liver Mφs are the largest population of resident Mφs in the body, and these individuals were isolated via Percoll gradient centrifugation. Approximately 95% of the cells were F4/80^+, indicating that the Mφs were of high purity (Fig. [95]S2). Nine samples were analyzed via transcriptome sequencing. Three triplicate libraries were established and sequenced to a depth of 36 million to 48 million reads. In total, 1129 DEGs were significantly different between the EV and ENV groups and the control group; these genes included 423 upregulated genes and 706 downregulated genes (Fig. [96]3A, B). We subsequently performed bioinformatics analyses of these DEGs. GO functional enrichment analysis revealed that the screened DEGs were involved mainly in the inflammatory response, immune system processes, signal transduction, the innate immune response, cell adhesion, etc. (Fig. [97]3C & Table [98]S3). KEGG analysis revealed that the DEGs were enriched mainly in the c-type lectin receptor signaling pathway, cytokine‒cytokine receptor interaction, neutrophil extracellular trap formation, the tumor necrosis factor (TNF) signaling pathway, the nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathway, the calcium signaling pathway, and the toll-like receptor signaling pathway (Fig. [99]3D-F & Table [100]S3). Fig. 3. [101]Fig. 3 [102]Open in a new tab Transcriptome sequencing was performed to identify differentially expressed genes (DEGs) and the biological pathways involved in the phagocytosis of nanovesicles (NVs, including EVs and ENVs). Mice were treated with PBS, EVs or ENVs (n = 3). After 4 h, liver macrophages (Mφs) were isolated and subjected to transcriptome analysis. (A) Statistics of the DEGs; the red column represents the number of upregulated genes, and the green column indicates the number of downregulated genes. (B) Venn diagrams showing the number of genes that were commonly and uniquely changed. (C) Heatmap of more than 1500 annotated genes expressed in liver Mφs after stimulation with PBS or NVs (Table [103]S2). The upregulated mRNAs in the treated group with respect to those in the control group are represented in red, and the downregulated mRNAs are presented in blue. (D) KEGG pathway enrichment analysis of the DEGs. (E-G) Gene Ontology analysis. Gene Ontology analysis of the downregulated and upregulated biological processes (E), cellular components (F) and molecular functions (G) related to the DEGs The TLR2 pathway plays important roles in the process of phagocytosis Eighteen genes of interest were selected from the prominent pathways detected by KEGG analysis, and the expression levels of these genes were validated with RT‒PCR; the data indicated that 7 of these 18 candidate genes were stably expressed at higher levels in the NVs-treated Mφs than that in the control Mφs (Fig. [104]4A). These genes included C-C motif chemokine receptor-like 2 (Cclr2), haptoglobin (Hp), hydroxycarboxylic acid receptor 2 (Hcar2), integrin beta 3 (Itgb3), the tumor necrosis factor receptor superfamily, member 1b (Tnfrsf1b), platelet-activating factor receptor (Ptafr), and toll-like receptor 2 (TLR2). Three siRNAs were designed for each of the 7 genes mentioned above, the interference effects were verified through RT‒PCR, and the most efficient siRNAs were selected for the subsequent experiments (Fig. [105]4B). siRNAs targeting these 7 genes were transfected into Mφs, after which endocytosis of the PKH67-labeled NVs was observed. Compared with those in the control and NC groups, treatment with siTLR2 markedly attenuated the uptake of NVs (EVs and ENVs) by Mφs (Fig. [106]4C). A relatively weak suppressive effect was also observed in the group treated with siTNFR, whereas no significant differences were observed in the groups transfected with other siRNAs (not shown). Next, the levels of IL-6 and TNF-α in the supernatant were detected by ELISA, as shown in Fig. [107]4D & E. The stimulation of EVs or ENVs strongly induced the production of IL-6 (33.04 ± 4.864 vs. 310.4 ± 22.35; 330.9 ± 24.09 pg/ml) and TNF-α (19.28 ± 2.153 vs. 228.0 ± 8.028, 234.1 ± 9.681 pg/ml) in Mφs, and siTLR2 significantly suppressed the secretion of IL-6 and TNF-α by EVs or ENVs. Furthermore, the activity of the TLR2 signaling pathway was detected via western blot. As expected, the administration of EVs or ENVs clearly increased the expression of TLR2 and MyD88 and promoted the phosphorylation of MAPK and NF-кB in Mφs (Fig. [108]4F). In brief, the TLR2/MyD88/MAPK/NF-кB pathway signaling pathway plays a key role in the phagocytosis of NVs by Mφ. Fig. 4. [109]Fig. 4 [110]Open in a new tab The TLR2 signaling pathway was demonstrated to be involved in the phagocytosis of NVs by Mφs. (A) Eighteen genes of interest were selected from the prominent pathways that were detected via KEGG analysis, and their expression levels were validated via qRT‒PCR. GAPDH served as an endogenous control. A total of 7/18 candidate genes were more highly expressed in the Mφs treated with NVs than that in the control (*** p < 0.001). (B) Three siRNAs were designed for each of the 7 DEGs, and the silencing efficiency was verified via qRT‒PCR. The most efficient siRNAs were selected for the subsequent experiments (*p < 0.05, **p < 0.01, *** p < 0.001). (C) siTLR2 significantly inhibited the uptake of NVs by liver Mφs. Mφs were transfected with the 7 different siRNAs or siNC and then treated with PKH67-labeled EVs or ENVs, after which the engulfment of NVs was observed. Compared with siNC and other siRNAs, siTNF-α and siTLR2 displayed suppressive effect, and the other siRNAs failed to suppress phagocytosis; these results are not presented. The scale bar represents 100 μm (*p < 0.05, **p < 0.01, *** p < 0.001). (D & E) siTLR2 downregulated the secretion of IL-6 (D) and TNF-α (E) by Mφs. The IL-6 and TNF-α levels in the medium of the Mφs mentioned in (C) were examined via ELISA (*** or ### p < 0.001). (F) Exposure to NVs activated the TLR2/MAPK/NF-κB pathway in liver Mφs. Isolated Mφs were stimulated with 40 µg of EVs or ENVs for 4 h. Then, proteins were extracted for western blot analysis (*** or ### p < 0.001) SpB suppressed phagocytosis in vitro SpB, which is extracted from a Chinese herb, is a selective receptor inhibitor of TLR2 and TLR4. To further test the above experimental results, Mφs were preadministered with SpB (200 nM) before being exposed to PKH67-labeled EVs or ENVs. Compared with those in control groups, the fluorescence signals in the NC group were not significantly different, whereas the intensity of the green signal was dramatically lower in the SpB-treated group (Fig. [111]5A). Consistent with the results of the cell experiments, SpB noticeably attenuated the production of IL-6 (Fig. [112]5B) and TNF-α (Fig. [113]5C) and decreased the phosphorylation of proteins in the MAPK and NF-кB pathways (Fig. [114]5D). In brief, SpB suppressed the phagocytic effect of Mφs on NVs in vitro by antagonizing TLR2/MyD88/MAPK/NF-кB signaling. Fig. 5. Fig. 5 [115]Open in a new tab Pretreatment with SpB significantly blocked the uptake of NVs by Mφs in vitro. Liver Mφs were treated with SpB (200 nM) or oil/DMSO for 30 min and then cultured with EVs or ENVs for 4 h. The untreated the Mφs were served as control group. (A) SpB significantly blocked the uptake of PKH67-labeled NVs by liver Mφs. The scale bar represents 100 μm (*p < 0.05, **p < 0.01, *** p < 0.001). (B & C) SpB strongly suppressed the secretion of IL-6 (B) and TNF-α (C) by liver Mφs (*** or ### p < 0.001). (D) SpB markedly inhibited the activation of the TLR2/MAPK/NF-κB pathway by liver Mφs (** or ## p < 0.01, *** or ### p < 0.001) ENVs-DOX was prepared and showed enhanced antitumor effects As the biological characteristics of NVs are similar but the yield of ENVs is greater, ENVs were chosen for the following experiments. DOX was subsequently successfully encapsulated in ENVs via an ultrasound method (Fig. [116]6A). As shown in Fig. [117]6B, the delivery efficiency improved with increasing DOX concentration within a certain range. When incubated with DOX at concentrations of 500 µg/ml, the delivery efficiency was 20.30 ± 0.85%. Increasing the concentration of DOX slightly improved the loading efficiency. According to these results, the optimal incubation concentration of DOX was 500 µg/ml, and 1 µg of ENVs (protein) contained 0.20 ± 0.01 µg of DOX (Fig. [118]6B). As depicted in Fig. [119]6C, the release behavior of DOX from the ENVs-DOX was determined at pH 7.4 to simulate physiological conditions and pH 5.0 to mimic the acid conditions in the cytolysosome. DOX exhibited fast release under both pH conditions during the first 10 h, and slightly greater DOX release was observed at the acidic pH value compared to that at the physiological pH value at later time points. In addition, the average size of the ENVs after loading DOX (174.6 nm) was larger than that before loading (127.7 nm), as shown in Fig. [120]6D. Fig. 6. [121]Fig. 6 [122]Open in a new tab The combination of doxorubicin (DOX) and ENVs generated enhanced antitumor effects in vitro. (A) Process diagram for the preparation of ENVs-DOX. DOX was encapsulated in ENVs via an ultrasound method. (B) 500 µg ENVs were incubated with different concentration of DOX, the loading efficiency of DOX were determined (*** p < 0.001). (C) DOX was released in vitro in PBS at pH 5.0 and 7.4(*** p < 0.001). (D) Hydrodynamic size distribution of ENVs after DOX loading. (E) The passive uptake of free DOX, ENVs and ENVs-DOX by PANC-1 cells. The scale bar represents 100 μm. In order to observe the DOX signal, the excitation filter/emission filter = 475 ~ 490/580 ~ 620 nm, for the PKH-67 signal, the excitation filter/emission filter = 475 ~ 490/505 ~ 535 nm (*** p < 0.001). (F) Cell viability was determined with a CCK-8 assay. PANC-1 cells were treated with different concentrations of DOX, ENVs or ENVs-DOX (n = 5, ***p < 0.001) The uptake of free DOX, PKH67-ENVs and PKH67-ENVs-DOX was detected via fluorescence imaging, and stronger red fluorescence (DOX) was detected in the cytoplasm of PANC-1 cells dealed with ENVs-DOX than in that of PANC-1 cells exposed to free DOX (Fig. [123]6E). Next, the outcomes of the CCK-8 assay revealed that cell viability was more efficiently inhibited by ENVs-DOX than by free DOX, and the IC50 values of ENVs-DOX and free DOX were 0.391 and 4.651 µM (p < 0.001), respectively, whereas the blank-ENVs slightly promoted the growth of PANC-1 cells (Fig. [124]6F). Taken together, these findings demonstrated that ENVs encapsulate DOX and promote the internalization of DOX by PANC-1 cells and that sufficient DOX concentrations can be released from ENVs to permit cancer cell death. SpB suppressed phagocytosis and enhanced the efficiency of ENVs delivery in vivo To determine the optimal time and concentration for the application of SpB in mice, SpB was dissolved in corn oil and then administered through intraperitoneal injection. After pretreatment with SpB, DiR-ENVs were injected, and fluorescent signals were detected. As shown in Fig. [125]S3, when SpB was administered 40 min in advance, the fluorescence intensities of the mouse body and liver were the weakest; this inhibitory effect could not be achieved when SpB was administered too early or too late. When the mice were exposed to different concentrations of SpB, the fluorescence signals in the liver decreased with increasing SpB concentrations. However, when the concentration of SpB exceeded 24 mg/kg, the suppressive effect did not increase significantly. As a result, SpB was administered 40 min in advance at a concentration of 24 mg/kg. As shown in Fig. [126]7A, after the subcutaneous tumor model was established, the mice received different treatments. Notably, compared with the oil/DMSO treatment group, the SpB + ENVs-DOX group presented a reduced accumulation of ENVs-DOX in nontarget organs (Fig. [127]7B & C), especially in the liver (54.52 ± 4.74 vs. 30.63 ± 2.20 × 10^6 p/s, p = 0.001; Fig. [128]7D & E). Moreover, pretreatment with SpB extended the retention time of ENVs-DOX in the blood circulation (Fig. [129]S4). Consequently, the distribution of the targeted nanocarriers in the tumor tissue of the SpB + ENVs-DOX group was approximately 2.57 times greater than that in the oil/DMSO + ENVs-DOX group (0.63 ± 0.07 vs. 1.62 ± 0.47 × 10^6 p/s, p = 0.023; Fig. [130]7D & E). As shown in Fig. [131]7F, G & H, tumor growth in the SpB + ENVs-DOX group was the slowest, followed by that in the oil/DMSO + ENVs-DOX group, and tumors in the group treated with blank ENVs grew slightly faster than those in the control group did. In summary, this study confirmed that SpB suppressed phagocytosis and enhanced the efficiency of ENV delivery at the animal level. Fig. 7. [132]Fig. 7 [133]Open in a new tab Antitumor efficacy of ENVs-DOX in vivo. (A) Schematic illustration of the tumor model establishment and therapeutic process of ENVs-DOX. Group information: (1) oil/DMSO + PBS as a control; (2) oil/DMSO + free DOX; (3) SpB + PBS; (4) oil/DMSO + blank ENVs; (5) oil/DMSO + ENVs-DOX; (6) SpB + ENVs-DOX. 200 µl oil/DMSO or SpB solution was administered through intraperitoneal injection; 40 min later, blank ENVs, DOX or ENVs-DOX were injected via the caudal vein. (B & C) SpB inhibited the non-targeted aggregation of ENVs-DOX (DiR-labeled) in vivo. Tumor-bearing nude mice (n = 3) were pretreated with or without SpB and injected with ENVs-DOX; then, the mice were subjected to live imaging at different time points (*p < 0.05, **p < 0.01, *** p < 0.001). (D & E) SpB promoted the accumulation of ENVs-DOX (DiR-labeled) in tumors. 6 h after injection of DiR/ENVs-DOX, those mice were sacrificed and the enriched DiR signals in the mice mentioned in (B) were detected (*p < 0.05, **p < 0.01, *** p < 0.001). (F) Body weight curves and tumor volume growth curves (G) of the mice (n = 5) (*p < 0.05). (H) Photographs of excised tumors from the mice in each group. Scale: 1 unit = 1 cm (*p < 0.05, **p < 0.01, *** p < 0.001) Evaluating the systemic toxicity of SpB and ENVs-DOX Histological and biochemical analyses were performed to evaluate the potential toxicity of SpB and ENVs-DOX to the important organs, such as the lung, liver, spleen, heart, and kidneys. As shown in Fig. [134]8A and Fig. [135]S5, the analyses of the liver, spleen and kidney in the experimental groups did not reveal histological alterations compared with those in the control group. However, the heart pathological images of the group treated with free DOX revealed more vacuolization of cardiomyocytes (indicated by arrows), which indicates the degeneration of muscle fibers. Moreover, fewer vacuolization areas were detected in the cardiomyocytes from the group treated with ENVs-DOX than in those from the free DOX-treated group. In addition, no obvious histological changes in heart sections were detected between the groups treated with SpB or blank ENVs and the control group. As presented in Fig. [136]8B, compared with the oil/DMSO + PBS group, free DOX injection markedly increased the ALT (95.26 ± 16.13 vs. 120.7 ± 9.70 U/l, p = 0.017) and CK-MB (28.34 ± 2.86 vs. 46.31 ± 8.72 U/l, p = 0.002352) levels, indicating that the group exhibited liver and cardiac damage, which may be related to DOX hepatotoxicity and cardiotoxicity. In contrast, ENVs-DOX exhibited lower toxicity to the liver and heart. The ALT and CK-MB levels in the SpB + ENVs-DOX groups did not increase significantly. The levels of CK-MB and ALT did not significantly differ between the other treatment groups and control group. In addition, there was no significant differences in the creatinine (CRE) levels were detected between the experimental groups and control group. Taken together, the ALT and CK-MB levels in the serum of the SpB + ENVs-DOX group were lower than those in the free DOX group, suggesting that enclosing DOX with nanovesicles and pretreatment with SpB could effectively reduce the toxicity of DOX. In addition, we also conducted experiments in C57BL/6 mice with normal immunity and found that SpB can also inhibit phagocytosis (30.11 ± 11.29 vs. 17.07 ± 3.45 × 10^6 p/s, p = 0.022; Fig. [137]S6). Furthermore, to clarify whether repeated administration of SpB affects the number and function of Mφs in mice, the densities of Mφs in the lungs, liver and spleen were assessed via immunohistochemical staining, and the serum LPS levels were detected via ELISA; however, the differences were not significant (Fig. [138]8C & D). Fig. 8. [139]Fig. 8 [140]Open in a new tab Evaluating the systemic toxicity of SpB and ENVs-DOX. (A) The histological Sect. (40×) of the organs (liver, spleen, kidney and heart) from the control, SpB and SpB + ENVs-DOX treated groups. Scale bar is 40 μm. The H-E staining of the organs from other groups could be seen in Figure [141]S5. Arrows indicate areas of vacuolization. (B) The test results of CRE, ALT and CK-MB from different groups (n = 5). Statistically analysis was performed using student’s t test and compared to the control group (*p < 0.05, **p < 0.01). (C) F4/80staining of the organs (lung, liver and spleen, 40×) from the control and SpB treated groups. Macrophages were stained with F4/80-specific antibodies (Brown). Scale bar is 40 μm. (D) Serum levels of LPS treated with control and SpB treated groups (n = 5). Data are presented as mean ± SD. No significant difference was found Discussion At present, chemical treatment is an important method for treating malignant tumors, but chemotherapy often has severe side effects. Many targeted drug carriers have been developed to improve therapeutic efficacy and reduce unpleasant side effects [[142]28, [143]29]. Owing to their nanoscale size and high biocompatibility, NVs have shown great potential for clinical application and have attracted much attention from scientists [[144]1–[145]5]. However, similar to other nanocarriers, NVs are rapidly cleared by the mononuclear phagocyte system during cyclic administration, and the half-life of NVs is estimated to be 30 min or less, which severely reduces their delivery efficiency [[146]9–[147]11]. Many studies have confirmed that NVs are predominantly removed by Mφs in the liver and spleen, but the specific mechanism involved has not been elucidated [[148]9–[149]11]. Previous articles have confirmed that the similarity of membrane proteins and smRNAs between ENVs and EVs is high, while the generation efficiency of ENVs is approximately 10 ~ 100 times greater than that of EVs. Therefore, ENVs are good alternatives to EVs as drug delivery nanocarriers [[150]6, [151]7]. In this study, we prepared and characterized ENVs and EVs derived from PANC-1 cells. The results confirmed that the morphology and characterization of ENVs and EVs are very similar and that the production of ENVs is approximately 25 times greater than that of EVs in the same PANC-1 cell population (Fig. [152]1). Next, we tracked the metabolic distribution of ENVs and EVs in vivo and unexpectedly found that fluorescence signals rapidly accumulated within the liver and spleen after intravenous injection, further confirming that ENVs and EVs were engulfed by Mφs; these findings are consistent with previous research results (Fig. [153]2 & Fig. [154]S1) [[155]9–[156]11]. We subsequently investigated the DEGs by comparing the gene transcription of treated and untreated Mφs. RAW264.7 cells or peritoneal Mφs were used in previous studies for phagocytosis experiments. We believe that primary macrophages better reflect the real status of Mφs. In recent years, single-cell and spatial transcriptomic technologies have helped us identify the heterogeneity of liver Mφs [[157]30–[158]33]. Therefore, the use of panmacrophage markers (such as F4/80 or CD68) to identify Mφs (the most abundant group of liver-resident Mφs) is imprecise [[159]33]. Liver Mφs were isolated via the recommended protocols and analyzed via immunofluorescence (Fig. [160]S2) [[161]25]. Liver Mφs were purified from mice treated with PBS, EVs, or ENVs and then subjected to transcriptome analysis. Through data analysis, we identified DEGs and screened approximately 18 genes of interest via pathway analysis (Fig. [162]3). RT‒PCR and siRNA interference experiments were also conducted for validation, and the data indicated that the TLR2 pathway plays a crucial role in phagocytosis (Fig. [163]4). Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that include TLR1-11 and play indispensable roles in innate immune responses. Among these receptors, TLR2 is the most versatile because it can recognize a wide range of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) [[164]34, [165]35]. siTLR2 significantly inhibited the uptake of both NVs by Mφs and inhibited the expression of TLR2 pathway-related proteins and the secretion of IL-6 and TNF-α (Fig. [166]5) [[167]36]. We chose to encapsulate DOX with ENVs for the subsequent experiments because their biological characteristics are similar to those of EVs, and the production of ENVs is greater than that of other vesicles (Fig. [168]1). Next, the DOX loading and release kinetics were explored. The results demonstrated that the optimal encapsulating ability was 0.20 ± 0.01 µg of DOX per 1 µg of ENVs at a DOX concentration of 500 µg/ml, and the enveloped DOX could be released more easily from ENVs in the cellular lysosomes (acid conditions) (Fig. [169]6). ENVs can encapsulate DOX through many methods, and the encapsulation efficiency of the same method varies greatly among different experiments [[170]37–[171]40]. In our study, we found that the efficiency of the coincubation method was too low. We ultimately applied ultrasound to encapsulate DOX, and the encapsulation efficiency of this method was significantly greater than that of the coincubation method. As anticipated, ENVs loaded with DOX promoted the uptake of DOX by PANC-1 cells and reduced the IC50 of DOX (Fig. [172]6). The pathway underlying the phagocytosis of nanovesicles by macrophages has been investigated previously, and numerous methods have been developed to inhibit phagocytosis; however, these methods are somewhat impractical or inconvenient [[173]23, [174]41, [175]42]. There are two main directions for preventing liver macrophage uptake [[176]23, [177]41, [178]42]. First, the NVs are modified or decorated [[179]43]. For example, Lili Cheng et al. constructed CD47-overexpressing NVs for photothermal therapy; these NVs suppressed Mφ-mediated phagocytosis by blocking CD47-SIRPα signaling and achieved enhanced accumulation at tumor sites [[180]21]. Second, macrophages can be killed, inhibited or saturated. Phosphate liposomes or gadolinium are commonly used to efficiently and quickly clear Mφs and can significantly prolong the circulation half-life of NVs [[181]10, [182]14]. However, approximately 2 weeks are needed for Mφs to gradually recover to normal numbers after phosphate liposomes or gadolinium are applied. Mφs are important components of the body’s innate immune system, and completely clearing Mφs may increase the risk of infection [[183]44]. In addition, administering high doses of autologous EVs can increase the rate of macrophage uptake [[184]14]. However, isolating and reinfusing autologous EVs is laborious and potentially risky. Overall, short-term inhibition of macrophage function is an optimal choice. SpB is a new type of anthracene compound that was discovered in the Chinese herb Scirpus yagara [[185]45]. Studies have shown that SpB is a low-toxicity selective antagonist of TLR2 and TLR4 that exhibits potent anti-inflammatory and antitumor effects and regulates the gut microbiota [[186]46, [187]47]. SpB is a water-insoluble reagent that was first dissolved in DMSO, diluted with corn oil, and then injected intraperitoneally in this study [[188]48]. DMSO and corn oil are biosafe solvents, and mice are not impacted by long-term administration of these agents intraperitoneally at recommended doses. Our results showed that administering SpB 30 ~ 40 min in advance may maximize its inhibitory effect (Fig. [189]S3), which may be related to the pharmacokinetics of SpB. Although the pharmacokinetics of SpB administered intraperitoneally have not been studied, Nuo-Shu Zou et al. demonstrated that SpB is rapidly eliminated within 30 min after intravenous injection at a dose of 0.5 mg/kg (half-life: 1.68 ± 0.73 h) [[190]49]. When the drug is administered too early or too late, an effective blood drug concentration may not be achieved. The inhibitory effect was positively correlated with the dose of SpB, but the suppressive effect did not increase significantly when the concentration of SpB exceeded 24 mg/kg. Finally, as shown in Fig. [191]7 & Fig. [192]S4, pretreatment with SpB prominently decreased the enrichment of ENVs-DOX in the liver in vivo and prolonged its half-life in the circulation. As expected, 2.57 times more ENVs-DOX accumulated in the tumor than in the untreated group. As a consequence, enhanced antitumor effects were observed in the SpB + ENVs-DOX group. Moreover, another study showed that SpB can curb the progression of pancreatic cancer cells and melanoma [[193]36, [194]50]. However, no significant antitumor effects were detected in this study, which may be caused by differences in the dosage or frequency of SpB administration. Notably, more ENVs accumulated in the lungs after treatment with SpB (Fig. [195]7). Similar phenomena have been reported in other studies, where enrichment in the lungs also increased after the depletion of macrophages in the body [[196]14]. These phenomena suggests that other cells in the body in addition to macrophages are involved in the uptake and elimination of NVs, and that the pharmacokinetics of NVs in vivo are more complicated than previously appreciated. Overall, Mφs in the liver and spleen are the main cells that take up and clear extracellular vesicles. Inhibiting or clearing liver and spleen Mφs can significantly prolong the circulation time of vesicle carriers (Fig. [197]7 & Fig. [198]S4) [[199]10, [200]14]. Evaluating the biocompatibility and toxicity of the prepared nanoparticles and SpB in vivo is vital [[201]26, [202]27]. Blank ENVs or SpB have no significant effect on the structure or function of the liver, kidney or heart. Significantly higher levels of ALT and CK-MB and obvious vacuolar degeneration of cardiomyocytes were observed in the mice that received free DOX, which was consistent with previous research [[203]26, [204]27]. The data showed that enclosing the DOX into ENVs could attenuate the toxicity of DOX, and pretreatment with SpB could further weaken the damage to the liver and heart associated with DOX, which may be due to the improved distribution of ENVs-DOX in the body (Fig. [205]8 & Fig. [206]S5). SpB is a well-known TLR2 and TLR4 receptor inhibitor, but its long-term effects on the number and function of Mφs are unknown. As shown in Fig. [207]8, the continuous administration of SpB did not affect the population size or immune response of Mφs. In addition, another DEG, TNFR, participates in phagocytosis, and siTNFR inhibited this process in vitro (data not shown). However, no antiphagocytic effects were observed when zafirlukast or R-7050 (which are both inhibitors of TNFR) was applied to the mice. This study has several limitations. First, NVs derived from human PANC-1 cells are natural mouse antigens that may activate the innate immune system. Therefore, the results of this study may not be applicable to vesicular carriers that are isolated from the same source. In addition, SpB displayed low toxicity and good safety in mice in our study and other studies. The short half-life of SpB prevents long-term suppression of macrophages in mice. However, its effectiveness and safety in the human body must be clarified through rigorous clinical trials. Finally, even the application of high-dose SpB could not completely inhibit the uptake of NVs by macrophages, which implies that other cells or signaling pathways are also involved, and further research is needed. Conclusions In summary, we demonstrated that the TLR2/MyD88/MAPK/NF-кB pathway plays a critical role in the uptake of NVs by Mφs, while SpB significantly blocked the TLR2-mediated clearance of NVs by Mφs both in vitro and in vivo, providing a novel and practical strategy for achieving better therapeutic outcomes in nanomedicine. Electronic supplementary material Below is the link to the electronic supplementary material. [208]Supplementary Material 1^ (21.4KB, docx) [209]Supplementary Material 2^ (39.4KB, xlsx) [210]12951_2024_3001_MOESM3_ESM.jpg^ (2.3MB, jpg) Supplementary Material 3: Figure S1. The representative immunofluorescence images of free DiI and DiI-labeled EVs group and Colocalization analysis [211]12951_2024_3001_MOESM4_ESM.jpg^ (986.7KB, jpg) Supplementary Material 4: Figure S2. Isolation and characterization of liver Mφs [212]12951_2024_3001_MOESM5_ESM.jpg^ (11.3MB, jpg) Supplementary Material 5: Figure S3. Exploring the optimal dosing interval and dosage of SpB in vivo [213]12951_2024_3001_MOESM6_ESM.jpg^ (251.8KB, jpg) Supplementary Material 6: Figure S4. SpB prolonged the residence time of ENVs in the peripheral blood [214]12951_2024_3001_MOESM7_ESM.jpg^ (2.6MB, jpg) Supplementary Material 7: Figure S5. Evaluating the systemic toxicity ENVs-DOX [215]12951_2024_3001_MOESM8_ESM.jpg^ (537.4KB, jpg) Supplementary Material 8: Figure S6. SpB suppressed the phagocytosis effect in immune normal mice Acknowledgements