Exosomes/MoS2 complex for targeting and effective photothermal therapy

Exosomes/MoS2 complex for targeting and effective photothermal therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exosomes/MoS2 complex for targeting and effective photothermal therapy Liyan Wang, Huizhi Chen, Haiyan Qiu, Zhenyu Xie, Shah Zada, Jianbo Sun, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6226702/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted 13 You are reading this latest preprint version Abstract Photothermal therapy (PTT) has been an attractive tumor treatment strategy in recent years. Two-dimensional molybdenum disulfide (MoS 2 )-based nanomaterials with high photothermal efficiency is a critical candidate for PTT. However, the tumor-targeting capability extremely needs to be improved for effective tumor treatment. In this work, we combine the MoS 2 nanodots with exosomes, native vesicles secreted from living cells, to construct a novel exosomes/MoS 2 complex (MoS 2 @ME) for effective tumor-targeted PTT. Through ultrasonic self-assembly membranes, MoS 2 nanodots are incorporated into MCF-7 exosomes. Similar to free MoS 2 , MoS 2 @ME shows significant photothermal conversion effect, causing nearly 100% necrosis proportion of MCF-7 and 4T1 cells under 1064 nm laser irradiation within 5 min (0.4 W/cm 2 ) in vitro . In particular, MoS 2 @ME presents noteworthy affinity for tumor cells, and in vivo studies further prove that it could accumulate at the tumor site efficiently. After intravenous injection with MoS 2 @ME plus NIR irradiation, the temperature of tumor site in 4T1 tumor-bearing mice could reach 46°C within a short time (~ 2 min). Notably, with the prolongation of NIR irradiation time, the temperature of tumors gradually increases and reaches the maximum temperature (52.3°C) at 8 min, which is far higher than that in the free MoS 2 group. More importantly, PTT using MoS 2 @ME exhibits much more effective antitumor therapy, as the tumor volume and tumor weight of mice in the MoS 2 @ME group are significantly lower than those in the PBS and MoS 2 groups ( P < 0.05), and even the tumor disappears completely. In vitro and in vivo studies demonstrate that the MoS 2 @ME shows excellent targeting capacity and photothermal effect, achieving effective photothermal cancer therapy. This work is expected to overcome the shortcomings of some photothermal materials, aiming to improve safety and effectiveness. The exosome-incorporated design strategy may pave a new way for molybdenum-based tumor-targeted PTT. Photothermal therapy Targeting Exosomes MoS2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Photothermal therapy (PTT), as an emerging tumor treatment method, has become attractive due to its high efficiency, spatiotemporally control, low side effects, and low cost[ 1 – 4 ]. In PTT, photothermal agents (PTAs) are critical components that can convert visible or near-infrared (NIR) light into heat at specific sites and raise the local temperature of the tumor, thereby achieving thermal ablation of solid tumors. In particular, NIR (800–1700 nm) light is applied frequently as the excitation light source owing to its strong penetrability and few side effects[ 5 – 9 ]. PTAs based on two-dimensional (2D) materials have gained increasing attention due to their ultra-thin structure, high specific surface area and unique photoelectric properties, showing great application potential [ 10 – 13 ]. Especially, 2D molybdenum disulfide (MoS 2 ) has been widely developed owing to its high biocompatibility, easy modification and strong NIR absorption[ 14 , 15 ]. It has been reported that small-sized 2D MoS 2 nanodots possess high photothermal conversion efficiency under NIR laser irradiation and excellent in vivo metabolic capacity without long-term safety concerns[ 16 ]. However, the small size also means ease of clearance by the mononuclear phagocyte system (MPS)[ 17 , 18 ], resulting in a short residence time at the lesion site. In addition, studies have shown that only no more than 5% small-sized nanomaterials could reach the tumor tissue to exert therapeutic effects successfully after intravenous injection[ 19 , 20 ]. It can be seen that there are certain challenges in passively targeted delivery of MoS 2 nanodots to target cells. Therefore, it is vital to improve the tumor targeting and enrichment ability of 2D MoS 2 nanodots for achieving efficient PTT. Nowadays, multiple lipid vesicles such as liposomes, exosomes, micelles, platelets, and red cell membranes, etc. have been extensively employed to construct PTA delivery systems thanks to their natural remarkable biocompatibility and in vivo stability[ 21 – 23 ]. Among them, exosomes, as biological vesicles secreted from cells with a size ranging from 30 to 150 nm[ 24 ], have broad prospects in the field of tumor-targeted PTA delivery owning to their unique high stability, biocompatibility and intrinsic homing/targeting properties. They are widely found in various biological liquid media such as blood, urine, saliva, milk, and cell culture fluid[ 25 ], containing a variety of biological macromolecules (e.g., lipids, proteins and nucleic acids) and carrying a large amount of information about donor cells[ 26 ]. For example, Wang et al. prepared exosome-like particles by irradiating the parental cells preloaded with Bi 2 Se 3 and DOX into the buds, which achieved excellent tumor homing and killing abilities through the combination of PTT and chemotherapy[ 27 ]. Liu et al. co-encapsulated serum exosomes derived from hyperthermia-treated mice with black phosphorus to prepare a novel cancer nano-vaccine, which showed long-term photothermal performance and immunomodulatory capabilities[ 28 ]. In view of this, the combination of exosomes and MoS 2 is a simple and effective way to achieve effective tumor-targeted PTT, which has not been reported in-depth so far. Herein, given the advantages of endogenous tumor exosomes and the characteristics of MoS 2 nanodots, we successfully construct an exosome-based PTA system for effective tumor-targeted photothermal cancer therapy. Specifically, with ultrasonic self-assembly of membranes, MoS 2 nanodots prepared by liquid exfoliation are incorporated into MCF-7 exosomes to prepare the exosome-based PTA system (MoS 2 @ME). Under 1064 nm laser irradiation within 5 min (0.4 W/cm 2 ), MoS 2 @ME could rise by 30℃ sharply, showing significant photothermal conversion effect (similar to that of 30 ppm MoS 2 ). In vitro experiments show that MCF-7 cells and 4T1 cells are almost 100% killed after treatment with MoS 2 @ME plus NIR irradiation. Moreover, MoS 2 @ME exhibits a higher uptake of tumor MCF-7 cells than normal MCF-10A cells owning to the selectivity of exosomes towards tumor cells. In vivo assays demonstrate that MoS 2 @ME could gradually accumulate at tumor site within 48 h. More importantly, the temperature of tumor site could rise to the lowest temperature of tumor ablation (46°C) within less than 2 min and reach the maximum temperature (52.3°C) at 8 min, resulting in a tumor growth inhibition rate of 75.92%. This work is expected to overcome the shortcomings of some photothermal materials such as poor targeting and difficulty in modification with biological macromolecules, thereby improving safety and effectiveness and developing a new model of tumor photothermal treatment based on novel photothermal materials. 2. Materials and methods 2.1 Materials Total Exosome Isolation Kit (from cell culture media), CD63 Monoclonal Antibody (for Western blot), Human CD81 Flow Detection Reagent were purchased from ThermoFisher Scientific, USA. 100 kDa Ultra Centrifugal Filters were purchased from Merck, Germany. BCA Protein Assay Kit (BCA), Live/Dead Viability/Cytotoxicity Assay Kit, PKH26, and Hoechst 33342 were purchased from Beyotime Biotechnology, China. Cell Counting Kit-8 (CCK-8) was purchased from Zetalife, Japan. CD81 antibody was purchased from Santa Cruz Biotechnology. PE Mouse Anti-Human CD81 and FITC Mouse Anti-Human CD63 were purchased from BD medical technology company. 2.2 Cell culture Human breast cancer cell (MCF-7) was cultured in DMEM complete medium containing 10% fetal bovine serum (Gibco, USA) and 1% Penicillin-Streptomycin solution (Gibco, USA). Human breast epithelial cell (MCF-10A) (purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Company) was cultured in its special medium (Zhong Qiao Xin Zhou, China). Mouse breast carcinoma cell (4T1) and mouse fibroblast cell (L929) were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% Penicillin-Streptomycin solution. All cell lines were incubated in humidified atmosphere of 5% CO 2 at 37°C. 2.3 Isolation of exosomes Firstly, exosome-depleted FBS was obtained by ultracentrifugation (Beckman, Optima XPN, USA) at 12000 × g , 4°C overnight. Then the exosome-depleted FBS was added into the 1% antibiotic/DMEM as the maintenance medium. When the confluence of MCF-7 cells reached about 90%, the original culture medium was replaced with maintenance medium, and the cells were placed in the CO 2 incubator for 48–72 h. After that, the supernatant was collected and centrifugated at 300 × g for 10 min, 2000 × g for 10 min and 10000 × g for 30 min in succession. Next, the resulting solution was mixed with the total exosome isolation kit at 4°C overnight and centrifuged at 10000 × g for 30 min. The pellet was washed with PBS using 100 kDa ultra centrifugal filters and resuspended in PBS to obtain MCF-7 cell exosomes (ME). 2.4 Characterization of exosomes The morphology of exosomes was analyzed by transmission electron microscopy (TEM; Hitachi, HT-7700, Japan). The size distribution of exosomes was measured by dynamic light scattering (DLS; Horiba, SZ-100, Japan) and nanoparticle tracking analysis (NTA; Particle Metrix, ZetaView, Germany). The protein concentration was obtained by BCA protein assay kit. Meanwhile, the exosomal marker proteins such as CD81 and CD63, were identified by Western blot and Flow cytometry (BD, FACS Canto II, USA). 2.5 Synthesis and characterization of MoS 2 Commercial MoS 2 bulk crystal (2 g) was placed into a stainless steel grinding jar, and vibrated at a frequency of 40 Hz for 4 h in an argon glove box. The grounded MoS 2 powder (200 mg) was added into a glass vial (20 mL), then 10 mL of n -butyllithium solution (2.5 M in cyclohexane) was carefully added to the vial slowly. The vial was quickly sealed and stored in a glove box for 2 days. Upon completion of the removal of the upper solution of n -butyllithium, the material must be washed with hexane three times and then sonicated with a water bath and a probe in 100 mL of water for 30 min each. After high-speed centrifugation (14800 r/min) for 10 min to remove large-sized MoS 2 , polyvinyl pyrrolidone (PVP, Mw = 58000) was added to the suspension and stirred overnight at room temperature. PVP-modified MoS 2 (PVP-MoS 2 ) nanodots were obtained after washing with deionized water from Millipore ultrafiltration tube and then stored at 4℃ before used. The primary physical properties of MoS 2 were characterized by TEM and XPS. TEM images and XPS spectra of MoS 2 were recorded on a transmission electron microscope (JEM-2100F, Japan) and a K-ALPHA (Thermo, USA) X-ray photoelectron spectroscopy. To determine the optimal processing conditions (material concentration and laser power), MoS 2 with different concentrations were exposed to 1064 nm NIR (CNIlaser, MILN-1064, China) for 10 min (0.4 W/cm 2 ). Additionally, 30 ppm of MoS 2 was subjected to different NIR laser powers for 10 min. As for the photothermal stability, 30 ppm MoS 2 was exposed to 1064 nm NIR light for 5 min (0.4 W/cm 2 ) and then cooled to room temperature. The two steps were repeated 5 times, and the temperature changes during this process were monitored by an Infrared Thermal Imaging Camera (FOTRIC, 625C-L25, China). 2.6 Preparation and characterization of MoS 2 @ME The exosomes (1×10 10 particles/mL) were mixed with 30 ppm MoS 2 and sonicated in the ice bath sonicator for 30 min to obtain MoS 2 -loaded exosomes (MoS 2 @ME, stored at 4℃). Its characterization methods were the same as that of exosomes and MoS 2 . Additionally, the investigation of its storage stabilities including protein, particle size, potential, and photothermal properties was performed via SDS-PAGE, DLS and Infrared Thermal Imaging Camera at day 0, day 1, day 3, day 5 and day 7, respectively. 2.7 In vitro investigation 2.7.1 Cell viability and toxicity The cell viability was determined by CCK-8 assay. Simply, MCF-7 (2×10 4 cells/mL) and 4T1 (1×10 5 cells/mL) cells were seeded into 48-well plates for 24 h, respectively. Then, the original medium was replaced with MoS 2 or MoS 2 @ME. After 4 h incubation, the cells would be exposed to a NIR laser (1064 nm, 0.4 W/cm 2 ) for 5 min. Finally, the cells were washed with PBS twice, and the medium containing 10% CCK-8 was added for 30 min. As for the biosafety of MoS 2 @ME in normal cells, MCF-10A and L929 cells were seeded into 96-well plates with 2×10 4 cells/mL for 24 h, respectively. Then the supernatant was replaced with MoS 2 or MoS 2 @ME (30 ppm of MoS 2 ) for 4 h and evaluated by CCK-8 according to the protocol above. 2.7.2 Blood compatibility The blood compatibility of MoS 2 @ME was evaluated by hemolytic test. The blood was collected from mouse’s eyeballs, and then centrifuged at 3000 r/min for 5 min to obtain red blood cells. Subsequently, MoS 2 @ME was incubated with the red blood cells for 30 min at 37℃ and centrifuged at 4℃. The supernatant, whose absorption at 540 nm was measured by a Microplate Reader (Biotek, Synergy H1, USA) to calculate the hemolytic rate, while the pellet cells were fixed with 4% polymerization and observed by SEM (JEOL, JCM-6000PLUS, Japan). 2.7.3 Cell uptake Confocal laser scanning microscope (CLSM; Lecia, TCS SP8, Germany) and Flow cytometry (Sony, MA900, Japan) were employed to examine the cellular uptake of exosomes and study their cellular selectivity. Specific steps were as follows: MCF-7, 4T1 and MCF-10A were seeded in the confocal dishes with 5×10 4 cells for 24 h. The MCF-7 exosomes were marked with PKH26. Then the labeled exosomes were incubated with the cells for 1 h, 2 h, 4 h, and 6 h, respectively. After that, the cells were washed with PBS twice, fixed with 4% polymerization for 15 min, and incubated with Hoechst 33342 before being imaged using CLSM. As for Flow cytometry (FCM) detection, MCF-7, 4T1 and MCF-10A were seeded into 24-well plates with 1×10 5 cells per well for 24 h, respectively. Then the cells were incubated with exosomes and MoS 2 @ME (labeled with PKH26) for 1 h. Subsequently, the cells were subjected to centrifugation at 300 × g (4℃) for 5 min to prepare single-cell suspensions for FCM analysis. 2.8 Animal experiment 2.8.1 Establishment of tumor-bearing mouse model Female Balb/c mice aged at 4 weeks, from Si Pei Fu Biotechnology (Beijing, China), were injected with 4T1 cells (5×10 6 cells/mL, 200 µL) subcutaneously into the right flank and fed in an animal facility under 25℃ and 55% of humidity for 5 ~ 7 days until the tumor size reached 50 ~ 100 mm 3 . All the animal experimental protocols were approved by the Animal Research Committee of Guangdong Medical University. 2.8.2 Biodistribution of MoS 2 @ME Exosomes and MoS 2 @ME were labeled with dye DiR (1,1′-dioctadecyl-3,3,3′′,3′-tetramethylindotricarbocyanine iodide) and purified with 100 kDa Ultra centrifugal filters at 14000 × g and 4℃ for 10 min. When the tumor size of mouse model reached 50 mm 3 , DiR-labeled exosomes (ME-DiR) and MoS 2 @ME (MoS 2 @ME-DiR) were intravenously injected into the tumor-bearing mice for 0 h, 2 h, 6 h, 24 h, and 48 h, respectively, and monitored by IVIS Spectrum (PerkinElmer, IVIS Lumina Ⅲ, USA). Then the mice were sacrificed and their organs such as heart, liver, spleen, lung, kidney and tumor issue were picked out to image ex-vivo . 2.8.3 Photothermal effect of MoS 2 @ME on solid tumors When the tumor volume reached 100 mm 3 , the mice were divided into four groups and intravenously injected with the PBS (two groups), MoS 2 (8 mg/kg), and MoS 2 @ME (8 mg/kg of MoS 2 ), respectively. After 24 h, except for one PBS group (as control group), the other groups were exposed to an NIR laser (1064 nm, 0.48 W/cm 2 ) for 10 min. The temperature changes at the tumor site were monitored by an Infrared Thermal Imaging Camera (FOTRIC, 625C-L25, China) during the irradiation. After that, the tumor volume was recorded daily until 20 days (the tumor volume = tumor length × tumor width square/2). Next, the tumor tissue would be separated and weighed to investigate the tumor growth inhibition value (TGI). TGI = tumor weight/control group tumor weight × 100%. 2.8.4 Biosafety evaluation in vivo Normal mice at 4 weeks were randomly divided into three groups and injected with PBS, MoS 2 (8 mg/kg), MoS 2 @ME (8 mg/kg of MoS 2 ) through tail vein. Their body weight was checked at day 3, day 5, and day 7, and vital organs were stained by Hematoxylin-Eosin Staining (H&E; Servicebio, China) after one week. As for tumor-bearing mice, their blood serum from eyeball was took to analyze liver function and renal function such as alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CRE), etc. Meanwhile, tumor tissues and major organs (heart, liver, spleen, lung, kidney) were collected and stained with H&E to assess the therapeutic effect and biocompatibility of MoS 2 @ME. 2.9 Statistical analysis All data was plotted as the mean ± S.D. and analyzed by Origin 2019b. One-way ANOVA was employed to compare the data of each group, and P < 0.05 was considered to indicate statistically significant. 3. Results 3.1 Preparation and characterization of MoS 2 and MoS 2 @ME The ultra-small MoS 2 nanodots were synthesized through lithium intercalation, followed by ultrasonic treatment. As shown in Figure S1 A , TEM image of the prepared MoS 2 exhibits a uniform nanodot morphology with a size ranging from 3 to 5 nm. The high-resolution TEM images reveal that the nanodots display continuous lattice stripes with a lattice spacing of 0.21 nm corresponding to the (006) plane of the MoS 2 crystal ( Figure S1 B ). In addition, the XPS survey spectra of MoS 2 nanodots display characteristic peaks attributed to Mo and S ( Figure S2 ), further confirming the successful preparation of MoS 2 nanodots. To improve the stability of MoS 2 nanodots in physiological environments, PVP was employed as a surface modifier to prepare the PVP-modified MoS 2 (MoS 2 -PVP) nanodots (represented by MoS 2 in this article). As shown in Figure S3A , even in a high salt concentration PBS solution, MoS 2 -PVP can still maintain the same dispersibility as in ultrapure water (UPW), indicating its good dispersion stability. Therefore, considering its biological application, MoS 2 -PVP stored in PBS was chosen for subsequent experiments. The exosomes (ME) from MCF-7 cell culture medium were isolated by polyethylene glycol precipitation (Total Exosome Isolation Kit), and the total protein concentration of exosomes measured by BCA was 14.86 mg/mL, indicating that this method can efficiently extract exosomes from cells. TEM image ( Figure S4A ) showed that the exosomes had a typical "cup-and-disc" shape. DLS analysis ( Figure S4B ) revealed that the particle size distribution of exosomes was concentrated with an average particle size of 102.5 ± 2.5 nm. Meanwhile, NTA analysis ( Figure S4C ) showed that the particle concentration of exosomes was 2.0×10 11 particles/mL, indicating that the obtained exosome had excellent quality. The Western Blot and Flow Cytometry results ( Figures S5A and S5B ) suggested that the exosomes had relatively abundant marker proteins CD63 and CD81. The above results implied that MCF-7 exosomes were successfully prepared, and could be used for subsequent experiments such as tumor targeted modification. After the successful preparation of ME, MoS 2 nanodots were incorporated into ME to prepare MoS 2 @ME complex through ultrasonic self-assembly membranes. Similar to MoS 2 , MoS 2 @ME solution presented a brown-black color (Fig. 2 A), with an obvious vesicle structure the same as MCF-7 exosomes (Fig. 2 B). DLS showed that the size of MoS 2 @ME was 90.8 ± 6.1 nm, which was slightly smaller than that of exosomes before loading (Fig. 2 C). The above results preliminarily suggested that MoS 2 has been loaded into exosomes. Likewise, NTA showed that the average particle diameter of the MoS 2 @ME was 138.8 nm with a particle concentration was 3.0×10 11 particles/mL, indicating a concentrated particle distribution and high particle concentration, which can meet the requirements of subsequent experiments (Fig. 2 D). SDS-PAGE was employed to separate the proteins, as shown in Figure S5C , the protein composition of MoS 2 @ME was basically the same as that of exosomes, suggesting that the protein composition of exosomes was retained after MoS 2 loading. Flow cytometry further verified that the MoS 2 @ME contained relatively high levels of exosomal marker proteins CD63 and CD81 with the positive rates greater than 90% (Fig. 2 E). The above results indicated that neither MoS 2 incorporation nor ultrasonic treatment affected the key proteins of exosomes, implying that the MoS 2 @ME has the basis for targeting tumors. The photothermal properties of MoS 2 @ME were investigated. As shown in Fig. 2 F, its temperature-raising ability was almost the same as that of MoS 2 and significantly higher than that of the control group (PBS), which indicated that MoS 2 @ME retained the excellent photothermal performance of MoS 2 . According to the photothermal stability investigation, it was found that MoS 2 @ME could maintain its photothermal conversion ability after 5 “on-and-off” cycles of NIR irradiation (Fig. 2 G), suggesting that repeated laser irradiation had no significant effect on its photothermal properties. Additionally, the major proteins, particle size and photothermal conversion properties of MoS 2 @ME hardly changed after 7 days, suggesting its storage stability at 4℃ ( Figures S6 and S7 ). 3.2 In vitro investigation CLSM and flow cytometry were used to evaluate the uptake of exosomes in different cells. CLSM results (Fig. 3 A) showed that the uptake of exosomes in different cells was time-dependent. Before 4 h, the uptake of exosomes in MCF-7 (human cancer breast cells) was significantly higher than that in MCF-10A (normal human breast cells), suggesting the homologous targeting ability of exosomes from MCF-7. The uptake of MCF-7 exosomes in 4T1 (mouse breast carcinoma cells) was also evaluated. The result was similar to that of MCF-7 cells, suggesting that exosomes have certain tumor cell tropism and universality. In addition, the uptake of exosomes in each cell within 4 h did not change much compared with 6 h, indicating that the cells could basically complete the uptake after 4 h of administration. Consistent with the results of CLSM, FCM results showed that the positive rate of MCF-7 is more than twice that of MCF-10A after incubation with exosomes, indicating that MCF-7 exosomes can be taken up by more MCF-7 cells (Fig. 3 B). In addition, the MFI relative value (MFI ME /MFI Control ) of the MCF-10A group was 6.1, while that of the MCF-7 group was 15.7, which is more than 2.5 times that of the MCF-10A group, consistent with the positive rate results. These results could preliminarily verify that exosomes have certain tumor cell selectivity. Furthermore, FCM was used to investigate the difference in the uptake of MoS 2 @ME between tumor cells and normal cells under the same incubation time and exosome concentration. In Fig. 3 C, the uptake of MoS 2 @ME in MCF-7 cells was higher than that in normal MCF-10A cells, indicating that MoS 2 @ME containing MCF-7 exosomes could be taken up by more MCF-7 cells. In particular, the difference in the uptake of MoS 2 @ME between the two types of cells was smaller than that caused by exosomes alone, which may due to the fact that MoS 2 hinders the preferential recognition of exosome surface proteins by cancer cells. The above results can preliminarily verify that MoS 2 @ME could retain the tumor cell targeting of exosomes, but MoS 2 may partially weaken their targeting. Subsequently, the photothermal killing effect of MoS 2 @ME on MCF-7 and 4T1 cells was examined. Figure 3 D showed that in MCF-7 cells, exosomes alone did not show cell killing effect regardless of whether NIR irradiation was applied, and the cell viability was also increased under NIR irradiation, which may be because the mild heat generated by NIR irradiation at this power density is suitable for cell growth and can promote cell proliferation to some extent. However, after treatment with MoS 2 and MoS 2 @ME, the cell viability decreased significantly under NIR irradiation compared with those without NIR and other groups, and the difference was extremely statistically significant ( P < 0.001). The live/dead staining (Fig. 3 E) images verified the above results. Similar to MCF-7, MoS 2 and MoS 2 @ME under NIR irradiation could significantly affect the viability of 4T1 cells, and a large number of necrotic tumor cells could be observed, while the inhibition or death of 4T1 cells in the corresponding control group was not obvious. Taken together, MoS 2 @ME retained the light-triggered photothermal performance of MoS 2 , which could significantly kill human and mouse tumor cells. Then, the toxicity of MoS 2 @ME on MCF-10A and mouse fibroblast L929 was tested (Fig. 3 F), and the results showed that exosomes, MoS 2 and MoS 2 @ME had no obvious toxicity. The cell viability of normal cells after treatment with MoS 2 @ME was > 80%, indicating its excellent biocompatibility. In addition, the hemocompatibility of MoS 2 @ME was tested by hemolysis assay. It was found that the hemolysis rate of MoS 2 @ME was close to 0.00% (lower than the standard value of 5%). However, due to a large ultraviolet absorption overlap between MoS 2 and red blood cells at the detection wavelength that may affect the results of hemolysis test, we further observed the morphology of red blood cells under SEM ( Figure S8 ), and the results showed that red blood cells treated with MoS 2 @ME still remained biconcave disc shape, suggesting that MoS 2 @ME did not affect the normal morphology of red blood cells and had good blood compatibility. 3.3 Animal experiments 3.3.1 In vivo biodistribution of exosomes and MoS 2 @ME DiR is a lipophilic cell membrane dye characterized by a significant increase in fluorescence intensity after binding to cell membranes or cell membrane-like structure. After DiR labeling, exosomes and MoS 2 @ME were placed in a blank 96-well plate. As shown in Figure S9 , the free DiR is blue-green, while the exosomes are blue after DiR labeling, and there is no obvious change in the appearance of exosomes. The DiR-labelled exosomes (ME-DiR) showed obvious fluorescence, indicating that the exosomes successfully bound to the dye DiR. It should be noted that the DiR-labelled MoS 2 @ME (MoS 2 @ME-DiR) showed weak fluorescence, the same as free dye. The possible reason is that the black MoS 2 shield in MoS 2 @ME absorbs photons/energy from the DiR-labeled membrane structure, thus attenuating the fluorescence received by the instrument detector. Anyway, the above results indicated that the exosomes and MoS 2 @ME were successfully labeled with DiR and could be used for subsequent imaging of living animals. Each sample labeled with DiR was injected into the tail vein of mice, and the results are shown in Fig. 4 . Within 48 h, free DiR was mainly distributed in the liver without obvious tumor accumulation, while exosomes and MoS 2 @ME could accumulate at the tumor sites within 2 h, and the accumulation at the tumor sites rose up as time went on, indicating that exosomes and MoS 2 @ME had certain tumor targeting properties and could accumulate in the tumor gradually along with blood circulation. After 48 h, the mice were sacrificed, whose main organs and tumor tissues were taken for ex-vivo imaging. As shown in Figs. 4 B and 4 C, free DiR was mainly distributed in the liver and spleen as expected, which meant its clearance by the mononuclear phagocyte system. In addition to the obvious fluorescence observed in the liver and spleen, accumulation of ME and MoS 2 @ME can also be observed in the lung and tumor tissues. Given the nanoscale size of ME and MoS 2 @ME, the possible reason for their accumulation in lung tissue is that the ultrafiltration operation during purification causes the aggregation or shape changes of some particles, resulting in an increase in particle size, which may then be mechanically trapped by pulmonary capillaries. Nonetheless, exosomes and MoS 2 @ME retained accumulation at the tumor sites. On the other hand, the photothermal treatment in this study was triggered by local NIR. It should be difficult for the materials distributed in other parts to produce photothermal effects in the absence of NIR, which reflects the safety advantages of this system. Taken together, exosomes and MoS 2 @ME have certain tumor targeting properties in vivo and can be used for subsequent drug-targeted therapy. Meanwhile, local irradiation of the tumor site should be performed during subsequent photothermal treatment to reduce the damage to the lungs. 3.3.2 Investigation of the photothermal therapeutic effect of MoS 2 @ME on tumor During the photothermal treatment, the temperature changes of tumor sites in mice were monitored. In Fig. 5 A, MoS 2 and MoS 2 @ME had significant effects on elevating the temperature of tumor tissue compared with PBS in the control group. In particular, after the administration of MoS 2 @ME, the lowest temperature of tumor ablation (46°C) could be reached within about 2 min. With the prolongation of NIR irradiation time, the temperature of tumors gradually increased and reached the maximum temperature (52.3°C) at 8 min. Notably, the temperature of the tumor site in the MoS 2 @ME group was far higher than that in the naked MoS 2 group, implying that MoS 2 @ME could increase the accumulation of MoS 2 in the tumor site due to the tumor targeting property of exosomes. These above results indicated that MoS 2 and MoS 2 @ME had significant photothermal effects on solid tumors, laying the foundation for subsequent tumor ablation or growth inhibition. After the treatment, the tumor size and the appearance of mice was continuously monitored. It can be seen that no obvious abnormalities were found in other tissues except tumor tissue ( Figure S10A ), indicating the safety of the treatment to a certain extent. Within 2 days, the tumor tissues in the MoS 2 and MoS 2 @ME groups became black, indirectly reflecting the significant photothermal killing effect of MoS 2 and MoS 2 @ME. With the time passed by, the tumor size increased in all groups (Fig. 5 B). The degree of increase from large to small was PBS, PBS + NIR group, MoS 2 + NIR group, and MoS 2 @ME + NIR group. After 20 days, the tumor-bearing mice were sacrificed, and the tumor tissues were peeled off. As shown in Fig. 5 B, compared with other groups, the tumor volume of the MoS 2 @ME group was significantly reduced under NIR irradiation, and the tumors even completely disappeared. At the end point, compared with the untreated PBS group, the tumor volume of the MoS 2 @ME group decreased under NIR irradiation with statistical difference ( P < 0.05), suggesting that the photothermal treatment had certain antitumor effect (Fig. 5 C). Similar to the results of tumor volume, the photothermal treatment could significantly reduce tumor weight compared to other groups (Fig. 5 D), and the tumor growth inhibition rate could reach over 60%. In particular, the MoS 2 @ME group can achieve a tumor growth inhibition rate of 75.92% under NIR irradiation, suggesting that MoS 2 @ME has a stronger tumor killing effect. In addition, the survival curves ( Figure S10C ) showed that there was no significant difference in the survival function of mice in each group, indicating that the photothermal treatment was relatively mild and had no significant impact on the survival and longevity of mice. 3.3.3 Blood biochemical and histological tests after treatment Subsequently, to verify the safety of MoS 2 @ME, blood taken from the eyeballs of tumor-bearing mice after various treatments was collected to measure the serum liver function, kidney function and other biochemical indicators. As shown in Figure S11 , ALT and AST, as sensitive indicators of liver function damage, were in the normal range in each group, suggesting that MoS 2 and MoS 2 @ME may only accumulate in the liver without significant impact on its structure and function. Similarly, the BUN and CRE in each group were normal, suggesting that the kidneys of mice after various treatments were healthy without obvious abnormalities. Moreover, the histological results showed that no obvious pathological damage was found in the main organs of tumor-bearing mice after various treatments (Fig. 5 E). These results further indicated that MoS 2 @ME had good biological safety. In addition, H&E staining was performed on the tumor tissues of mice after various treatments (Fig. 5 E). It was found that compared with other groups, the tumor cells in MoS 2 @ME + NIR group were loosely arranged, the number of cells was significantly reduced, and the staining of the cytoplasm became lighter, suggesting the apoptosis and necrosis of tumor tissues, which confirmed the strong killing effect of MoS 2 @ME on solid tumors under NIR irradiation. 3.3.4 In vivo biosafety evaluation The in vivo safety of MoS 2 @ME was further investigated by monitoring the body weight of normal mice after tail vein administration and the appearance of major organs after one week ( Figures S12 and S13 ). As shown in Figure S12A , there was no significant change in the shape of each organ except that the liver was black in MoS 2 and MoS 2 @ME groups, likely from the color of MoS 2 . Within 7 days of administration of MoS 2 and MoS 2 @ME, the mice in each group survived with good health: smooth white fur, normal appetite, vigorous activities, and no significant change in body weight ( Figure S12B ). H&E staining ( Figure S13 ) showed that no obvious pathological changes were found in the organs. The results of biosafety evaluation in normal mice were consistent with those of tumor-bearing mice as expected (Fig. 5 E), indicating the excellent in vivo safety of MoS 2 and MoS 2 @ME. 4. Discussion Some nano-sized materials are easily cleared by the mononuclear phagocyte system (Mononuclear Phagocyte System, MPS). The current emerging drug delivery system-exosomes, as biological endogenous nanovesicles, are expected to avoid the aforementioned problems[ 29 ]. Studies have demonstrated the incorporation of exosomes and nanomaterials for biological applications such as wound healing[ 30 ]. Exosomes contain proteins such as CD63 and CD81, which are general biomarkers for biological characterization[ 31 ]. The micro structure, size distribution and concentration are important indicators for verifying the quality of exosome isolation[ 32 ]. The results of TEM and DLS showed that the MCF-7 exosomes extracted based on the polyethylene glycol precipitation method had a typical "cup-disc shape" with a size distribution of 101–105 nm, which was in line with the size range of 30–150 nm for exosomes. Western blotting and flow cytometry showed the MCF-7 exosomes were rich in tetraspanins CD63 and CD81. PTAs are a key component of photothermal therapy, which convert light energy into thermal effect, causing local protein denaturation and cell membrane damage in tumor tissue, and then inducing tumor cell apoptosis or necrosis[ 33 ]. In PTT, the heat generated by the PTAs is critical and directly determines the tumor therapeutic effect[ 34 ]. The 2D photothermal agent MoS 2 demonstrates attractive photothermal conversion efficiency[ 35 , 36 ]. When activated by NIR, the heating effect of MoS 2 is proportional to the concentration, laser power, and time; at a lower concentration of 30 ppm and a lower power of 0.4 W/cm 2 , the temperature rise of MoS 2 within 5 min is still greater than 32°C, which is obviously higher than that of the control group. At the same time, after 5 "on-off" cycles, MoS 2 still maintains similar heating and cooling rates (photothermal conversion efficiency). The above properties of MoS 2 potentially reduce the possibility of repeated drug administration in vivo . However, the lack of targeting and potential safety issue limits the in vivo application of MoS 2 [ 37 ]. In this regard, this study plans to employ endogenous exosomes as carriers to construct MoS 2 @ME complex, aiming to endowing targeting capacity. In view of the fact that the size, uniformity of distribution and surface properties of the preparation directly affect its distribution and metabolism in body tissues[ 23 ], this study conducted a series of investigations on the basic properties of MoS 2 @ME, including morphology, particle size, photothermal properties and protein ingredients. The consistence of MoS 2 @ME and MoS 2 on heating rate and heating and cooling cycle curves suggests that MoS 2 @ME retained the photothermal properties of MoS 2 . The MoS 2 @ME displayed concentrated particle size distribution (ranging from 85 to 112 nm) and retained protein components. The above results show that the preparation for MoS 2 delivery is successfully prepared. MoS 2 @ME involves two important elements of MoS 2 and exosomes in our design. Among them, since molybdenum is one of the essential trace elements for animals and humans[ 37 ], MoS 2 has high biocompatibility in 2D transition metal sulfide-based PTAs[ 14 , 38 ]. The exosomes naturally have good biocompatibility due to their cell membrane-like structures[ 39 ]. The toxicity of the above materials and their preparation were analyzed by CCK-8 and live/dead staining. The results showed that none of the above materials and preparation obviously affected cell survival in tumor cells or normal cell lines (cell viability > 80%, both in human and murine cells) in the absence of NIR, and the preparation did not cause hemolysis in red blood cells, indicating that the preparation retains the biocompatibility of the raw material and has considerable in vitro safety. At the same time, under NIR triggering, the preparation showed near 100% killing of MCF-7 and 4T1 cells, indicating that it has excellent light-triggered photothermal conversion ability and tumor killing effect. The above discovery had provided importation indication for the subsequent in vivo animal experiments. In addition, the exosomes used in the system theoretically have homologous targeting and can actively target tumor cells. Because the biogenesis of exosomes determines that they naturally carry a large number of donor cell information and cell type-specific proteins found in the parent cell membrane, the adhesion proteins on their surface have been shown to be parental cell-prone and can attach to target cells[ 40 ]. Qualitative and quantitative analysis by CLSM and flow cytometry found that the uptake of exosomes was related to incubation time and cell type. The cellular uptake of exosomes within 6 h was proportional to incubation time, and was basically saturated at 4 h. Compared with normal MCF-10A cells, tumor exosomes have higher cellular uptake in tumor MCF-7 and 4T1 cells; flow cytometry results show that the positive rate difference between MCF-7 and MCF-10A uptake can reach 60%. After incorporation of MoS 2 , this difference decreased, but still maintained a higher uptake by MCF-7 cells. The above results are basically in line with expectations, which preliminarily indicate that the preparation presents tumor cell selectivity. 4T1 subcutaneous xenograft tumor model was established for verification of in vivo biodistribution and photothermal therapy effects with Balb/c mice. The growth and metastasis characteristics of 4T1 cells are very similar to breast cancer in humans, and thus they are widely used as a model of human breast cancer[ 41 , 42 ]. With DiR labelling, the tissue distribution of the preparation in tumor-bearing mice was investigated. Mouse tail vein administration of DiR was used to rule out the possibility of accumulation of the dye itself in the tumor. However, semi-quantitative fluorescence showed that the mouse tumor site showed weaker fluorescence after administration of DiR than before administration. The possible reason is that the dye itself is prone to aggregation. In order to completely rule out the aggregation of DiR dye and its possible impact on the kinetics of the preparation, future experiments may consider directly modifying fluorescent molecules on exosomes/hybrid vesicles, or use emerging technologies such as photoacoustic imaging to eliminate their interference[ 43 , 44 ]. In vivo biodistribution results showed that as the carrier or preparation circulates in the blood, its fluorescence at the tumor site gradually increases, and the results are significantly different from the control group, indicating that the preparation has certain tumor targeting properties. However, compared with other major metabolic organs such as liver and spleen, the targeting ability is still not excellent. This can be explained by the results from some reports: when injected intravenously, some unmodified tumor-derived exosomes can be rapidly taken up by MPS in the liver and spleen[ 45 – 47 ]. In particular, some studies have shown that exosomes have higher tumor cell uptake than dye when injected intratumorally, suggesting that the targeting of exosomes is more likely to be reflected in the exchange of information with neighboring cells[ 48 ]. In view of this, in the future, modification of exosomes targeting ligands or intratumorally local injection can be considered to highlight their active targeting. In PTT, the light source is the "switch" that triggers its therapeutic effect, usually visible light (400–800 nm) or near-infrared light (NIR, 800–1700 nm). Compared with visible light, which can be absorbed by many endogenous substances in biological tissues and cause scattering[ 49 ], NIR's longer wavelength can reduce tissue scattering and increase penetration depth[ 50 ], so it is considered to be favored. However, current PTAs are mostly concentrated in the NIR region (NIR-I, 800–1000 nm), which still has limited penetration depth and heat resistance caused by thermal shock[ 51 ]. Taking this into consideration, our work employed 1064 nm laser as irradiation light source, which is in the second zone near-infrared light (NIR-II, 1000–1700 nm) and can achieve lower tissue scattering and deeper penetration into the skin tissue, thereby realizing improved therapeutic efficacy [ 52 ]. At the same time, in view of the complexity of the environment and metabolic processes of cells in animals compared with those in vitro , a power density of 0.48 W/cm 2 , which was slightly higher than that in cell experiments (0.4 W/cm 2 ), was finally selected for animal experiments and the laser irradiation time was extended. At 2 min, the temperature of tumor site treated with MoS 2 @ME has increased by more than 18℃, which was similar to the results of in vitro experiment, suggesting that the system has strong tissue penetration in a short time. This photothermal condition is still relatively mild, and no obvious skin damage was seen on tumor-bearing mice without PTAs, indicating that NIR-II at this power has good safety in normal tissues. When MoS 2 and MoS 2 @ME were administered, black scabs appeared at the tumor site of mice, reflecting the powerful lethality of the photothermal treatment. It has been reported that when tissue temperature rises to 42°C, irreversible tissue damage occurs; when it rises to 42–46°C, cell necrosis will occur within 10 minutes; and when the temperature reaches 46–52°C, cells will form microvascular thrombosis, local ischemia leads to rapid death[ 53 ]. It can be seen from the thermal imaging and heating curve of animal tumor sites that after treatment with MoS 2 and MoS 2 @ME, the temperature of tumor site can reach 46°C in a short period of time. It is worth noting that MoS 2 @ME group reaches the killing temperature faster than the MoS 2 group. The final tumor inhibition rate also confirmed this judgment. The MoS 2 @ME exhibited stronger inhibition of tumor growth than MoS 2 alone during the whole treatment periods. The results suggested that the strategy was simple but potent, which can achieve tumor photothermal ablation at lower laser power and in a shorter treatment course. For example, our preparation needed only single treatment with 0.48 W/cm 2 , while the MoS 2 coated with hyaluronic acid (HA) required 1 W/cm 2 and triple treatment[ 54 ]. In another case, MoS 2 modified with polyethylene glycol (PEG) and vanadium (V- MoS 2 @PEG) employed 808 nm laser (NIR-I, relatively week penetration) at 0.6 W/cm 2 for 4-times irradiation[ 55 ]. In addition, the study also examined the safety of the treatment from the perspectives of normal mice and tumor-bearing mice and their main organs. Body weight, histological staining, blood biochemistry and other indicators all showed that the treatment had no significant impact on the mice's daily activities and the structure and function of important organs. Based on the above, after intravenous injection of MoS 2 @ME for 24 h, the photothermal therapy mode exposing the tumor site to 0.48 W/cm 2 1064 nm NIR for 10 min has significant tumor killing effect and good safety in vivo . 5. Conclusion In this work, we have successfully prepared MoS 2 @ME for advanced photothermal tumor therapy. With the help of ultrasonic self-assembly membranes, MoS 2 nanodots were incorporated into MCF-7 exosomes. The obtained MoS 2 @ME efficiently killed MCF-7 and 4T1 cells under NIR irradiation. In the absence of NIR, it did not show toxicity to tumor cells MCF-7, 4T1 and normal cells MCF-10A, L929, indicating its good biocompatibility and in vitro safety. Moreover, MoS 2 @ME basically retained the tumor cell selectivity of exosomes and could achieve in vivo tumor targeting. Furthermore, it presented significant tumor tissue heating effects in 4T1 tumor-bearing mice as soon as the NIR was switched. Despite mild laser conditions, it showed significantly lower tumor volume and weight, and higher tumor growth inhibition rate than naked MoS 2 . In a word, the results of mice study, including tumor appearance, volume and weight, survival rate, body weight and major organ changes, showed that the delivery system based on MoS 2 and exosomes could achieve in vivo tumor targeting, efficient photothermal anti-tumor and certain biological safety. Generally, a new strategy of constructing biomaterials for PTT is demonstrated in this paper, which may provide a new insight for the development of PTT. Declarations Ethics and consent to participate declarations All the animal experimental protocols were approved by the Animal Research Committee of Guangdong Medical University. Conflict of Interest The authors declare no conflict of interest. Funding This work is supported by Guangdong Basic and Applied Basic Research Foundation (2021B1515140006), Special projects in key areas for general colleges and universities of Guangdong Province (2021ZDZX2061, 2024ZDZX2068), Featured Innovation Projects for General Colleges and Universities of Guangdong Province (2022KTSCX042), Medical Scientific Research Foundation of Guangdong Province (A2023241), Funds for PhD Researchers of Guangdong Medical University in 2023 (4SG23184G and 4SG23233G), Dongguan Social Development Technology Project-Key Project (20231800940842), Songshan Lake Science and Technology Correspondent Project (20234403-01KCJ-G), Construction Project of Nano Technology and Application Engineering Research Center of Guangdong Medical University (4SG24179G), College Students’ innovation and entrepreneurship training program (202410571010, S202410571059, S202410571061, S202410571054, 202310571001, 202310571004, 202310571024), College Students’ innovation experiment program of Guangdong Medical University (FYDM004), and Discipline Construction Project of Guangdong Medical University (1019K20220003). Author Contribution Liyan Wang: Writing–original draft, Methodology, Formal analysis, Data curation, Conceptualization. Huizhi Chen: Writing–original draft, Methodology, Formal analysis, Data curation. Haiyan Qiu: Investigation, Formal analysis, Data curation. Zhenyu Xie: Validation, Supervision. Shah Zada: Investigation, Formal analysis, Data curation. Jianbo Sun: Validation, Supervision. Chengyu Lu: Writing–review & editing, Resources, Project administration, Funding acquisition. Zhan Zhou: Writing–review & editing, Resources, Project administration, Funding acquisition. Xinsheng Peng: Funding acquisition, Project administration, Resources, Writing–review & editing. Ruizheng Liang: Project administration, Resources, Writing–review & editing. Yubin Zhou: Funding acquisition, Project administration, Resources, Writing–review & editing. Acknowledgements Not applicable. 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University","correspondingAuthor":false,"prefix":"","firstName":"Zhan","middleName":"","lastName":"Zhou","suffix":""},{"id":435670466,"identity":"3b972ce1-a9c3-4bad-8209-8fffa0eb323c","order_by":8,"name":"Xinsheng Peng","email":"","orcid":"","institution":"Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinsheng","middleName":"","lastName":"Peng","suffix":""},{"id":435670467,"identity":"6c9b9271-79eb-4774-b27f-fd0b3bca0ee9","order_by":9,"name":"Ruizheng Liang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACPmYILWcAoZkJa2GDqjEGamFsIE4LlE7cQLwWduZnD7/uqUvfLpH8/AFDhXViA/vZAwQcxmZuLPOMLXfnjDTDBoYz6YkNPHkJBLQwmElLHODJ3XAjh7GBse1wYoMEjwEBLezfgFok0g3AWv4RpYXHTPLDAYMEiJYG4rSUSTMcSDDc2fPMcEbCsXTjNp4c/Fr4+Y9vk/xxoE7enD35wYcPNday/exn8GsBAWYeGCuBARFTeAHjD2JUjYJRMApGwcgFAK+nPOxNGMFUAAAAAElFTkSuQmCC","orcid":"","institution":"Guangdong Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ruizheng","middleName":"","lastName":"Liang","suffix":""},{"id":435670468,"identity":"24c4a880-8fb8-4e78-b28b-34e3562c215b","order_by":10,"name":"Yubin Zhou","email":"","orcid":"","institution":"Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yubin","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-03-14 13:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6226702/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6226702/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-025-03665-8","type":"published","date":"2025-09-29T15:57:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79568831,"identity":"91eb07be-fcd7-4a01-b4a7-3915a5e0f0be","added_by":"auto","created_at":"2025-03-31 10:02:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1718139,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the preparation of MoS\u003csub\u003e2\u003c/sub\u003e@ME and its therapeutic mechanism for PTT.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6226702/v1/cec6dacfec39d75f14e5efe3.jpg"},{"id":79568830,"identity":"2046b69b-9e02-4b1b-bdc2-3558b61ad50e","added_by":"auto","created_at":"2025-03-31 10:02:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":989238,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization and photothermal properties of MoS\u003csub\u003e2\u003c/sub\u003e@ME. (A) The appearance of MoS\u003csub\u003e2\u003c/sub\u003e@ME. (B) The microscopic appearance of MoS\u003csub\u003e2\u003c/sub\u003e@ME (Scale bar=100 nm). (C) DLS assay result of MoS\u003csub\u003e2\u003c/sub\u003e@ME. (D) NTA assay result of MoS\u003csub\u003e2\u003c/sub\u003e@ME. (E) Flow cytometer measurement of the exosomal markers. (F) Photothermal performance of MoS\u003csub\u003e2\u003c/sub\u003e@ME. (G) Thermal cycling stability of MoS\u003csub\u003e2\u003c/sub\u003e@ME.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6226702/v1/c7ec982f841520165b3e3375.jpg"},{"id":79568347,"identity":"ab657b41-98ad-42bb-835a-741d8ea8a3a9","added_by":"auto","created_at":"2025-03-31 09:54:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1727815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e investigation of MoS\u003csub\u003e2\u003c/sub\u003e@ME. (A) CLSM detection of cellular uptake of MCF-7 exosomes (Scale bar = 25 μm). (B) FCM detection of cellular uptake of MCF-7 exosomes. (C) FCM detection of cellular uptake of MoS\u003csub\u003e2\u003c/sub\u003e@ME. (D) CCK-8 method to detect the tumor cell killing effect of MoS\u003csub\u003e2\u003c/sub\u003e@ME \u003cem\u003ein vitro\u003c/em\u003e, compared with the corresponding control group \"PBS+NIR\", *\u003cem\u003eP\u003c/em\u003e＜0.05, ***\u003cem\u003eP\u003c/em\u003e＜0.001. (E) Live/dead staining images of tumor cells after different treatments (Scale bar = 400 μm). (F) Biocompatibility of MoS\u003csub\u003e2\u003c/sub\u003e@ME in different cell lines.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6226702/v1/96c74a54020ff2d274c2e7c2.jpg"},{"id":79568836,"identity":"3426d85f-0fca-49b8-b453-9208bfb1634d","added_by":"auto","created_at":"2025-03-31 10:02:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1247443,"visible":true,"origin":"","legend":"\u003cp\u003eBiodistribution of MoS\u003csub\u003e2\u003c/sub\u003e@ME in tumor-bearing mice. (A) Fluorescence imaging of 4T1 tumor-bearing mice post administration. (B) Fluorescence imaging of MoS\u003csub\u003e2\u003c/sub\u003e@ME in different organs. (C) Semi-quantitative results of the fluorescence imaging.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6226702/v1/8746c6ff85689e830a57ed9f.jpg"},{"id":79569418,"identity":"5a72a437-c4fb-4a88-80f7-0879614126d0","added_by":"auto","created_at":"2025-03-31 10:10:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2418274,"visible":true,"origin":"","legend":"\u003cp\u003ePhotothermal therapy of tumor-bearing mice (\u003cem\u003en=4\u003c/em\u003e). (A) Thermal imaging of mice and the thermal curve in the tumor site. (B) Appearance of tumors in tumor-bearing mice during treatment (left) and \u003cem\u003eex-vitro \u003c/em\u003e(right). (C) Tumor volume change, (D) tumor weight and tumor inhibition rate of mice after different treatments. (E) H\u0026amp;E staining of the main organs and tumor tissues after different treatments (Scale bar = 100 μm). *\u003cem\u003eP\u003c/em\u003e＜0.05, **\u003cem\u003eP\u003c/em\u003e＜0.01, ***\u003cem\u003eP\u003c/em\u003e＜0.001. I: PBS; II: PBS+NIR; III: MoS\u003csub\u003e2\u003c/sub\u003e+NIR; IV: MoS\u003csub\u003e2\u003c/sub\u003e@ME+NIR.\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6226702/v1/cfbbce9af7ae24ca86915c10.jpg"},{"id":92883689,"identity":"5464a96e-eec1-43e8-baa5-2b1b1d62c00f","added_by":"auto","created_at":"2025-10-06 16:07:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11645260,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6226702/v1/375fe27d-6115-41db-b6d6-e06614247f31.pdf"},{"id":79568833,"identity":"c96c7231-b871-44b2-80e7-c181b00542c4","added_by":"auto","created_at":"2025-03-31 10:02:05","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6366486,"visible":true,"origin":"","legend":"","description":"","filename":"ExosomesMoS2photothermaltherapySI20250314.docx","url":"https://assets-eu.researchsquare.com/files/rs-6226702/v1/e8b9187dd9238e9cb22d4aff.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosomes/MoS2 complex for targeting and effective photothermal therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePhotothermal therapy (PTT), as an emerging tumor treatment method, has become attractive due to its high efficiency, spatiotemporally control, low side effects, and low cost[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In PTT, photothermal agents (PTAs) are critical components that can convert visible or near-infrared (NIR) light into heat at specific sites and raise the local temperature of the tumor, thereby achieving thermal ablation of solid tumors. In particular, NIR (800\u0026ndash;1700 nm) light is applied frequently as the excitation light source owing to its strong penetrability and few side effects[\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. PTAs based on two-dimensional (2D) materials have gained increasing attention due to their ultra-thin structure, high specific surface area and unique photoelectric properties, showing great application potential [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Especially, 2D molybdenum disulfide (MoS\u003csub\u003e2\u003c/sub\u003e) has been widely developed owing to its high biocompatibility, easy modification and strong NIR absorption[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It has been reported that small-sized 2D MoS\u003csub\u003e2\u003c/sub\u003e nanodots possess high photothermal conversion efficiency under NIR laser irradiation and excellent \u003cem\u003ein vivo\u003c/em\u003e metabolic capacity without long-term safety concerns[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the small size also means ease of clearance by the mononuclear phagocyte system (MPS)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], resulting in a short residence time at the lesion site. In addition, studies have shown that only no more than 5% small-sized nanomaterials could reach the tumor tissue to exert therapeutic effects successfully after intravenous injection[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It can be seen that there are certain challenges in passively targeted delivery of MoS\u003csub\u003e2\u003c/sub\u003e nanodots to target cells. Therefore, it is vital to improve the tumor targeting and enrichment ability of 2D MoS\u003csub\u003e2\u003c/sub\u003e nanodots for achieving efficient PTT.\u003c/p\u003e \u003cp\u003eNowadays, multiple lipid vesicles such as liposomes, exosomes, micelles, platelets, and red cell membranes, etc. have been extensively employed to construct PTA delivery systems thanks to their natural remarkable biocompatibility and \u003cem\u003ein vivo\u003c/em\u003e stability[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Among them, exosomes, as biological vesicles secreted from cells with a size ranging from 30 to 150 nm[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], have broad prospects in the field of tumor-targeted PTA delivery owning to their unique high stability, biocompatibility and intrinsic homing/targeting properties. They are widely found in various biological liquid media such as blood, urine, saliva, milk, and cell culture fluid[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], containing a variety of biological macromolecules (e.g., lipids, proteins and nucleic acids) and carrying a large amount of information about donor cells[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. For example, Wang et al. prepared exosome-like particles by irradiating the parental cells preloaded with Bi\u003csub\u003e2\u003c/sub\u003eSe\u003csub\u003e3\u003c/sub\u003e and DOX into the buds, which achieved excellent tumor homing and killing abilities through the combination of PTT and chemotherapy[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Liu et al. co-encapsulated serum exosomes derived from hyperthermia-treated mice with black phosphorus to prepare a novel cancer nano-vaccine, which showed long-term photothermal performance and immunomodulatory capabilities[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In view of this, the combination of exosomes and MoS\u003csub\u003e2\u003c/sub\u003e is a simple and effective way to achieve effective tumor-targeted PTT, which has not been reported in-depth so far.\u003c/p\u003e \u003cp\u003eHerein, given the advantages of endogenous tumor exosomes and the characteristics of MoS\u003csub\u003e2\u003c/sub\u003e nanodots, we successfully construct an exosome-based PTA system for effective tumor-targeted photothermal cancer therapy. Specifically, with ultrasonic self-assembly of membranes, MoS\u003csub\u003e2\u003c/sub\u003e nanodots prepared by liquid exfoliation are incorporated into MCF-7 exosomes to prepare the exosome-based PTA system (MoS\u003csub\u003e2\u003c/sub\u003e@ME). Under 1064 nm laser irradiation within 5 min (0.4 W/cm\u003csup\u003e2\u003c/sup\u003e), MoS\u003csub\u003e2\u003c/sub\u003e@ME could rise by 30℃ sharply, showing significant photothermal conversion effect (similar to that of 30 ppm MoS\u003csub\u003e2\u003c/sub\u003e). \u003cem\u003eIn vitro\u003c/em\u003e experiments show that MCF-7 cells and 4T1 cells are almost 100% killed after treatment with MoS\u003csub\u003e2\u003c/sub\u003e@ME plus NIR irradiation. Moreover, MoS\u003csub\u003e2\u003c/sub\u003e@ME exhibits a higher uptake of tumor MCF-7 cells than normal MCF-10A cells owning to the selectivity of exosomes towards tumor cells. \u003cem\u003eIn vivo\u003c/em\u003e assays demonstrate that MoS\u003csub\u003e2\u003c/sub\u003e@ME could gradually accumulate at tumor site within 48 h. More importantly, the temperature of tumor site could rise to the lowest temperature of tumor ablation (46\u0026deg;C) within less than 2 min and reach the maximum temperature (52.3\u0026deg;C) at 8 min, resulting in a tumor growth inhibition rate of 75.92%. This work is expected to overcome the shortcomings of some photothermal materials such as poor targeting and difficulty in modification with biological macromolecules, thereby improving safety and effectiveness and developing a new model of tumor photothermal treatment based on novel photothermal materials.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eTotal Exosome Isolation Kit (from cell culture media), CD63 Monoclonal Antibody (for Western blot), Human CD81 Flow Detection Reagent were purchased from ThermoFisher Scientific, USA. 100 kDa Ultra Centrifugal Filters were purchased from Merck, Germany. BCA Protein Assay Kit (BCA), Live/Dead Viability/Cytotoxicity Assay Kit, PKH26, and Hoechst 33342 were purchased from Beyotime Biotechnology, China. Cell Counting Kit-8 (CCK-8) was purchased from Zetalife, Japan. CD81 antibody was purchased from Santa Cruz Biotechnology. PE Mouse Anti-Human CD81 and FITC Mouse Anti-Human CD63 were purchased from BD medical technology company.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell culture\u003c/h2\u003e \u003cp\u003eHuman breast cancer cell (MCF-7) was cultured in DMEM complete medium containing 10% fetal bovine serum (Gibco, USA) and 1% Penicillin-Streptomycin solution (Gibco, USA). Human breast epithelial cell (MCF-10A) (purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Company) was cultured in its special medium (Zhong Qiao Xin Zhou, China). Mouse breast carcinoma cell (4T1) and mouse fibroblast cell (L929) were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% Penicillin-Streptomycin solution. All cell lines were incubated in humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Isolation of exosomes\u003c/h2\u003e \u003cp\u003eFirstly, exosome-depleted FBS was obtained by ultracentrifugation (Beckman, Optima XPN, USA) at 12000 \u003cem\u003e\u0026times; g\u003c/em\u003e, 4\u0026deg;C overnight. Then the exosome-depleted FBS was added into the 1% antibiotic/DMEM as the maintenance medium. When the confluence of MCF-7 cells reached about 90%, the original culture medium was replaced with maintenance medium, and the cells were placed in the CO\u003csub\u003e2\u003c/sub\u003e incubator for 48\u0026ndash;72 h. After that, the supernatant was collected and centrifugated at 300 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min, 2000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min and 10000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 30 min in succession. Next, the resulting solution was mixed with the total exosome isolation kit at 4\u0026deg;C overnight and centrifuged at 10000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 30 min. The pellet was washed with PBS using 100 kDa ultra centrifugal filters and resuspended in PBS to obtain MCF-7 cell exosomes (ME).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Characterization of exosomes\u003c/h2\u003e \u003cp\u003eThe morphology of exosomes was analyzed by transmission electron microscopy (TEM; Hitachi, HT-7700, Japan). The size distribution of exosomes was measured by dynamic light scattering (DLS; Horiba, SZ-100, Japan) and nanoparticle tracking analysis (NTA; Particle Metrix, ZetaView, Germany). The protein concentration was obtained by BCA protein assay kit. Meanwhile, the exosomal marker proteins such as CD81 and CD63, were identified by Western blot and Flow cytometry (BD, FACS Canto II, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Synthesis and characterization of MoS\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eCommercial MoS\u003csub\u003e2\u003c/sub\u003e bulk crystal (2 g) was placed into a stainless steel grinding jar, and vibrated at a frequency of 40 Hz for 4 h in an argon glove box. The grounded MoS\u003csub\u003e2\u003c/sub\u003e powder (200 mg) was added into a glass vial (20 mL), then 10 mL of \u003cem\u003en\u003c/em\u003e-butyllithium solution (2.5 M in cyclohexane) was carefully added to the vial slowly. The vial was quickly sealed and stored in a glove box for 2 days. Upon completion of the removal of the upper solution of \u003cem\u003en\u003c/em\u003e-butyllithium, the material must be washed with hexane three times and then sonicated with a water bath and a probe in 100 mL of water for 30 min each. After high-speed centrifugation (14800 r/min) for 10 min to remove large-sized MoS\u003csub\u003e2\u003c/sub\u003e, polyvinyl pyrrolidone (PVP, Mw\u0026thinsp;=\u0026thinsp;58000) was added to the suspension and stirred overnight at room temperature. PVP-modified MoS\u003csub\u003e2\u003c/sub\u003e (PVP-MoS\u003csub\u003e2\u003c/sub\u003e) nanodots were obtained after washing with deionized water from Millipore ultrafiltration tube and then stored at 4℃ before used.\u003c/p\u003e \u003cp\u003eThe primary physical properties of MoS\u003csub\u003e2\u003c/sub\u003e were characterized by TEM and XPS. TEM images and XPS spectra of MoS\u003csub\u003e2\u003c/sub\u003e were recorded on a transmission electron microscope (JEM-2100F, Japan) and a K-ALPHA (Thermo, USA) X-ray photoelectron spectroscopy. To determine the optimal processing conditions (material concentration and laser power), MoS\u003csub\u003e2\u003c/sub\u003e with different concentrations were exposed to 1064 nm NIR (CNIlaser, MILN-1064, China) for 10 min (0.4 W/cm\u003csup\u003e2\u003c/sup\u003e). Additionally, 30 ppm of MoS\u003csub\u003e2\u003c/sub\u003e was subjected to different NIR laser powers for 10 min. As for the photothermal stability, 30 ppm MoS\u003csub\u003e2\u003c/sub\u003e was exposed to 1064 nm NIR light for 5 min (0.4 W/cm\u003csup\u003e2\u003c/sup\u003e) and then cooled to room temperature. The two steps were repeated 5 times, and the temperature changes during this process were monitored by an Infrared Thermal Imaging Camera (FOTRIC, 625C-L25, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Preparation and characterization of MoS\u003csub\u003e2\u003c/sub\u003e@ME\u003c/h2\u003e \u003cp\u003eThe exosomes (1\u0026times;10\u003csup\u003e10\u003c/sup\u003e particles/mL) were mixed with 30 ppm MoS\u003csub\u003e2\u003c/sub\u003e and sonicated in the ice bath sonicator for 30 min to obtain MoS\u003csub\u003e2\u003c/sub\u003e-loaded exosomes (MoS\u003csub\u003e2\u003c/sub\u003e@ME, stored at 4℃). Its characterization methods were the same as that of exosomes and MoS\u003csub\u003e2\u003c/sub\u003e. Additionally, the investigation of its storage stabilities including protein, particle size, potential, and photothermal properties was performed via SDS-PAGE, DLS and Infrared Thermal Imaging Camera at day 0, day 1, day 3, day 5 and day 7, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 \u003cem\u003eIn vitro\u003c/em\u003e investigation\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 Cell viability and toxicity\u003c/h2\u003e \u003cp\u003eThe cell viability was determined by CCK-8 assay. Simply, MCF-7 (2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL) and 4T1 (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL) cells were seeded into 48-well plates for 24 h, respectively. Then, the original medium was replaced with MoS\u003csub\u003e2\u003c/sub\u003e or MoS\u003csub\u003e2\u003c/sub\u003e@ME. After 4 h incubation, the cells would be exposed to a NIR laser (1064 nm, 0.4 W/cm\u003csup\u003e2\u003c/sup\u003e) for 5 min. Finally, the cells were washed with PBS twice, and the medium containing 10% CCK-8 was added for 30 min.\u003c/p\u003e \u003cp\u003eAs for the biosafety of MoS\u003csub\u003e2\u003c/sub\u003e@ME in normal cells, MCF-10A and L929 cells were seeded into 96-well plates with 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL for 24 h, respectively. Then the supernatant was replaced with MoS\u003csub\u003e2\u003c/sub\u003e or MoS\u003csub\u003e2\u003c/sub\u003e@ME (30 ppm of MoS\u003csub\u003e2\u003c/sub\u003e) for 4 h and evaluated by CCK-8 according to the protocol above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2 Blood compatibility\u003c/h2\u003e \u003cp\u003eThe blood compatibility of MoS\u003csub\u003e2\u003c/sub\u003e@ME was evaluated by hemolytic test. The blood was collected from mouse\u0026rsquo;s eyeballs, and then centrifuged at 3000 r/min for 5 min to obtain red blood cells. Subsequently, MoS\u003csub\u003e2\u003c/sub\u003e@ME was incubated with the red blood cells for 30 min at 37℃ and centrifuged at 4℃. The supernatant, whose absorption at 540 nm was measured by a Microplate Reader (Biotek, Synergy H1, USA) to calculate the hemolytic rate, while the pellet cells were fixed with 4% polymerization and observed by SEM (JEOL, JCM-6000PLUS, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3 Cell uptake\u003c/h2\u003e \u003cp\u003eConfocal laser scanning microscope (CLSM; Lecia, TCS SP8, Germany) and Flow cytometry (Sony, MA900, Japan) were employed to examine the cellular uptake of exosomes and study their cellular selectivity. Specific steps were as follows: MCF-7, 4T1 and MCF-10A were seeded in the confocal dishes with 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells for 24 h. The MCF-7 exosomes were marked with PKH26. Then the labeled exosomes were incubated with the cells for 1 h, 2 h, 4 h, and 6 h, respectively. After that, the cells were washed with PBS twice, fixed with 4% polymerization for 15 min, and incubated with Hoechst 33342 before being imaged using CLSM. As for Flow cytometry (FCM) detection, MCF-7, 4T1 and MCF-10A were seeded into 24-well plates with 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well for 24 h, respectively. Then the cells were incubated with exosomes and MoS\u003csub\u003e2\u003c/sub\u003e@ME (labeled with PKH26) for 1 h. Subsequently, the cells were subjected to centrifugation at 300 \u0026times; \u003cem\u003eg\u003c/em\u003e (4℃) for 5 min to prepare single-cell suspensions for FCM analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Animal experiment\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1 Establishment of tumor-bearing mouse model\u003c/h2\u003e \u003cp\u003eFemale Balb/c mice aged at 4 weeks, from Si Pei Fu Biotechnology (Beijing, China), were injected with 4T1 cells (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL, 200 \u0026micro;L) subcutaneously into the right flank and fed in an animal facility under 25℃ and 55% of humidity for 5\u0026thinsp;~\u0026thinsp;7 days until the tumor size reached 50\u0026thinsp;~\u0026thinsp;100 mm\u003csup\u003e3\u003c/sup\u003e. All the animal experimental protocols were approved by the Animal Research Committee of Guangdong Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 Biodistribution of MoS\u003csub\u003e2\u003c/sub\u003e@ME\u003c/h2\u003e \u003cp\u003eExosomes and MoS\u003csub\u003e2\u003c/sub\u003e@ME were labeled with dye DiR (1,1\u0026prime;-dioctadecyl-3,3,3\u0026prime;\u0026prime;,3\u0026prime;-tetramethylindotricarbocyanine iodide) and purified with 100 kDa Ultra centrifugal filters at 14000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4℃ for 10 min. When the tumor size of mouse model reached 50 mm\u003csup\u003e3\u003c/sup\u003e, DiR-labeled exosomes (ME-DiR) and MoS\u003csub\u003e2\u003c/sub\u003e@ME (MoS\u003csub\u003e2\u003c/sub\u003e@ME-DiR) were intravenously injected into the tumor-bearing mice for 0 h, 2 h, 6 h, 24 h, and 48 h, respectively, and monitored by IVIS Spectrum (PerkinElmer, IVIS Lumina Ⅲ, USA). Then the mice were sacrificed and their organs such as heart, liver, spleen, lung, kidney and tumor issue were picked out to image \u003cem\u003eex-vivo\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.8.3 Photothermal effect of MoS\u003csub\u003e2\u003c/sub\u003e@ME on solid tumors\u003c/h2\u003e \u003cp\u003eWhen the tumor volume reached 100 mm\u003csup\u003e3\u003c/sup\u003e, the mice were divided into four groups and intravenously injected with the PBS (two groups), MoS\u003csub\u003e2\u003c/sub\u003e (8 mg/kg), and MoS\u003csub\u003e2\u003c/sub\u003e@ME (8 mg/kg of MoS\u003csub\u003e2\u003c/sub\u003e), respectively. After 24 h, except for one PBS group (as control group), the other groups were exposed to an NIR laser (1064 nm, 0.48 W/cm\u003csup\u003e2\u003c/sup\u003e) for 10 min. The temperature changes at the tumor site were monitored by an Infrared Thermal Imaging Camera (FOTRIC, 625C-L25, China) during the irradiation. After that, the tumor volume was recorded daily until 20 days (the tumor volume\u0026thinsp;=\u0026thinsp;tumor length \u0026times; tumor width square/2). Next, the tumor tissue would be separated and weighed to investigate the tumor growth inhibition value (TGI). TGI\u0026thinsp;=\u0026thinsp;tumor weight/control group tumor weight \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.8.4 Biosafety evaluation \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eNormal mice at 4 weeks were randomly divided into three groups and injected with PBS, MoS\u003csub\u003e2\u003c/sub\u003e (8 mg/kg), MoS\u003csub\u003e2\u003c/sub\u003e@ME (8 mg/kg of MoS\u003csub\u003e2\u003c/sub\u003e) through tail vein. Their body weight was checked at day 3, day 5, and day 7, and vital organs were stained by Hematoxylin-Eosin Staining (H\u0026amp;E; Servicebio, China) after one week. As for tumor-bearing mice, their blood serum from eyeball was took to analyze liver function and renal function such as alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CRE), etc. Meanwhile, tumor tissues and major organs (heart, liver, spleen, lung, kidney) were collected and stained with H\u0026amp;E to assess the therapeutic effect and biocompatibility of MoS\u003csub\u003e2\u003c/sub\u003e@ME.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data was plotted as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. and analyzed by Origin 2019b. One-way ANOVA was employed to compare the data of each group, and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Preparation and characterization of MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME\u003c/h2\u003e \u003cp\u003eThe ultra-small MoS\u003csub\u003e2\u003c/sub\u003e nanodots were synthesized through lithium intercalation, followed by ultrasonic treatment. As shown in \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e, TEM image of the prepared MoS\u003csub\u003e2\u003c/sub\u003e exhibits a uniform nanodot morphology with a size ranging from 3 to 5 nm. The high-resolution TEM images reveal that the nanodots display continuous lattice stripes with a lattice spacing of 0.21 nm corresponding to the (006) plane of the MoS\u003csub\u003e2\u003c/sub\u003e crystal (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e). In addition, the XPS survey spectra of MoS\u003csub\u003e2\u003c/sub\u003e nanodots display characteristic peaks attributed to Mo and S (\u003cb\u003eFigure S2\u003c/b\u003e), further confirming the successful preparation of MoS\u003csub\u003e2\u003c/sub\u003e nanodots. To improve the stability of MoS\u003csub\u003e2\u003c/sub\u003e nanodots in physiological environments, PVP was employed as a surface modifier to prepare the PVP-modified MoS\u003csub\u003e2\u003c/sub\u003e (MoS\u003csub\u003e2\u003c/sub\u003e-PVP) nanodots (represented by MoS\u003csub\u003e2\u003c/sub\u003e in this article). As shown in \u003cb\u003eFigure S3A\u003c/b\u003e, even in a high salt concentration PBS solution, MoS\u003csub\u003e2\u003c/sub\u003e-PVP can still maintain the same dispersibility as in ultrapure water (UPW), indicating its good dispersion stability. Therefore, considering its biological application, MoS\u003csub\u003e2\u003c/sub\u003e-PVP stored in PBS was chosen for subsequent experiments.\u003c/p\u003e \u003cp\u003eThe exosomes (ME) from MCF-7 cell culture medium were isolated by polyethylene glycol precipitation (Total Exosome Isolation Kit), and the total protein concentration of exosomes measured by BCA was 14.86 mg/mL, indicating that this method can efficiently extract exosomes from cells. TEM image (\u003cb\u003eFigure S4A\u003c/b\u003e) showed that the exosomes had a typical \"cup-and-disc\" shape. DLS analysis (\u003cb\u003eFigure S4B\u003c/b\u003e) revealed that the particle size distribution of exosomes was concentrated with an average particle size of 102.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 nm. Meanwhile, NTA analysis (\u003cb\u003eFigure S4C\u003c/b\u003e) showed that the particle concentration of exosomes was 2.0\u0026times;10\u003csup\u003e11\u003c/sup\u003e particles/mL, indicating that the obtained exosome had excellent quality. The Western Blot and Flow Cytometry results (\u003cb\u003eFigures S5A and S5B\u003c/b\u003e) suggested that the exosomes had relatively abundant marker proteins CD63 and CD81. The above results implied that MCF-7 exosomes were successfully prepared, and could be used for subsequent experiments such as tumor targeted modification.\u003c/p\u003e \u003cp\u003eAfter the successful preparation of ME, MoS\u003csub\u003e2\u003c/sub\u003e nanodots were incorporated into ME to prepare MoS\u003csub\u003e2\u003c/sub\u003e@ME complex through ultrasonic self-assembly membranes. Similar to MoS\u003csub\u003e2\u003c/sub\u003e, MoS\u003csub\u003e2\u003c/sub\u003e@ME solution presented a brown-black color (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), with an obvious vesicle structure the same as MCF-7 exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). DLS showed that the size of MoS\u003csub\u003e2\u003c/sub\u003e@ME was 90.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1 nm, which was slightly smaller than that of exosomes before loading (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The above results preliminarily suggested that MoS\u003csub\u003e2\u003c/sub\u003e has been loaded into exosomes. Likewise, NTA showed that the average particle diameter of the MoS\u003csub\u003e2\u003c/sub\u003e@ME was 138.8 nm with a particle concentration was 3.0\u0026times;10\u003csup\u003e11\u003c/sup\u003e particles/mL, indicating a concentrated particle distribution and high particle concentration, which can meet the requirements of subsequent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). SDS-PAGE was employed to separate the proteins, as shown in \u003cb\u003eFigure S5C\u003c/b\u003e, the protein composition of MoS\u003csub\u003e2\u003c/sub\u003e@ME was basically the same as that of exosomes, suggesting that the protein composition of exosomes was retained after MoS\u003csub\u003e2\u003c/sub\u003e loading. Flow cytometry further verified that the MoS\u003csub\u003e2\u003c/sub\u003e@ME contained relatively high levels of exosomal marker proteins CD63 and CD81 with the positive rates greater than 90% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The above results indicated that neither MoS\u003csub\u003e2\u003c/sub\u003e incorporation nor ultrasonic treatment affected the key proteins of exosomes, implying that the MoS\u003csub\u003e2\u003c/sub\u003e@ME has the basis for targeting tumors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe photothermal properties of MoS\u003csub\u003e2\u003c/sub\u003e@ME were investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, its temperature-raising ability was almost the same as that of MoS\u003csub\u003e2\u003c/sub\u003e and significantly higher than that of the control group (PBS), which indicated that MoS\u003csub\u003e2\u003c/sub\u003e@ME retained the excellent photothermal performance of MoS\u003csub\u003e2\u003c/sub\u003e. According to the photothermal stability investigation, it was found that MoS\u003csub\u003e2\u003c/sub\u003e@ME could maintain its photothermal conversion ability after 5 \u0026ldquo;on-and-off\u0026rdquo; cycles of NIR irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), suggesting that repeated laser irradiation had no significant effect on its photothermal properties. Additionally, the major proteins, particle size and photothermal conversion properties of MoS\u003csub\u003e2\u003c/sub\u003e@ME hardly changed after 7 days, suggesting its storage stability at 4℃ (\u003cb\u003eFigures S6 and S7\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2 \u003cem\u003eIn vitro\u003c/em\u003e investigation\u003c/h2\u003e \u003cp\u003eCLSM and flow cytometry were used to evaluate the uptake of exosomes in different cells. CLSM results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) showed that the uptake of exosomes in different cells was time-dependent. Before 4 h, the uptake of exosomes in MCF-7 (human cancer breast cells) was significantly higher than that in MCF-10A (normal human breast cells), suggesting the homologous targeting ability of exosomes from MCF-7. The uptake of MCF-7 exosomes in 4T1 (mouse breast carcinoma cells) was also evaluated. The result was similar to that of MCF-7 cells, suggesting that exosomes have certain tumor cell tropism and universality. In addition, the uptake of exosomes in each cell within 4 h did not change much compared with 6 h, indicating that the cells could basically complete the uptake after 4 h of administration. Consistent with the results of CLSM, FCM results showed that the positive rate of MCF-7 is more than twice that of MCF-10A after incubation with exosomes, indicating that MCF-7 exosomes can be taken up by more MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In addition, the MFI relative value (MFI\u003csub\u003eME\u003c/sub\u003e/MFI\u003csub\u003eControl\u003c/sub\u003e) of the MCF-10A group was 6.1, while that of the MCF-7 group was 15.7, which is more than 2.5 times that of the MCF-10A group, consistent with the positive rate results. These results could preliminarily verify that exosomes have certain tumor cell selectivity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, FCM was used to investigate the difference in the uptake of MoS\u003csub\u003e2\u003c/sub\u003e@ME between tumor cells and normal cells under the same incubation time and exosome concentration. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the uptake of MoS\u003csub\u003e2\u003c/sub\u003e@ME in MCF-7 cells was higher than that in normal MCF-10A cells, indicating that MoS\u003csub\u003e2\u003c/sub\u003e@ME containing MCF-7 exosomes could be taken up by more MCF-7 cells. In particular, the difference in the uptake of MoS\u003csub\u003e2\u003c/sub\u003e@ME between the two types of cells was smaller than that caused by exosomes alone, which may due to the fact that MoS\u003csub\u003e2\u003c/sub\u003e hinders the preferential recognition of exosome surface proteins by cancer cells. The above results can preliminarily verify that MoS\u003csub\u003e2\u003c/sub\u003e@ME could retain the tumor cell targeting of exosomes, but MoS\u003csub\u003e2\u003c/sub\u003e may partially weaken their targeting.\u003c/p\u003e \u003cp\u003eSubsequently, the photothermal killing effect of MoS\u003csub\u003e2\u003c/sub\u003e@ME on MCF-7 and 4T1 cells was examined. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD showed that in MCF-7 cells, exosomes alone did not show cell killing effect regardless of whether NIR irradiation was applied, and the cell viability was also increased under NIR irradiation, which may be because the mild heat generated by NIR irradiation at this power density is suitable for cell growth and can promote cell proliferation to some extent. However, after treatment with MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME, the cell viability decreased significantly under NIR irradiation compared with those without NIR and other groups, and the difference was extremely statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The live/dead staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) images verified the above results. Similar to MCF-7, MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME under NIR irradiation could significantly affect the viability of 4T1 cells, and a large number of necrotic tumor cells could be observed, while the inhibition or death of 4T1 cells in the corresponding control group was not obvious. Taken together, MoS\u003csub\u003e2\u003c/sub\u003e@ME retained the light-triggered photothermal performance of MoS\u003csub\u003e2\u003c/sub\u003e, which could significantly kill human and mouse tumor cells.\u003c/p\u003e \u003cp\u003eThen, the toxicity of MoS\u003csub\u003e2\u003c/sub\u003e@ME on MCF-10A and mouse fibroblast L929 was tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), and the results showed that exosomes, MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME had no obvious toxicity. The cell viability of normal cells after treatment with MoS\u003csub\u003e2\u003c/sub\u003e@ME was \u0026gt;\u0026thinsp;80%, indicating its excellent biocompatibility. In addition, the hemocompatibility of MoS\u003csub\u003e2\u003c/sub\u003e@ME was tested by hemolysis assay. It was found that the hemolysis rate of MoS\u003csub\u003e2\u003c/sub\u003e@ME was close to 0.00% (lower than the standard value of 5%). However, due to a large ultraviolet absorption overlap between MoS\u003csub\u003e2\u003c/sub\u003e and red blood cells at the detection wavelength that may affect the results of hemolysis test, we further observed the morphology of red blood cells under SEM (\u003cb\u003eFigure S8\u003c/b\u003e), and the results showed that red blood cells treated with MoS\u003csub\u003e2\u003c/sub\u003e@ME still remained biconcave disc shape, suggesting that MoS\u003csub\u003e2\u003c/sub\u003e@ME did not affect the normal morphology of red blood cells and had good blood compatibility.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Animal experiments\u003c/h2\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 \u003cem\u003eIn vivo\u003c/em\u003e biodistribution of exosomes and MoS\u003csub\u003e2\u003c/sub\u003e@ME\u003c/h2\u003e \u003cp\u003eDiR is a lipophilic cell membrane dye characterized by a significant increase in fluorescence intensity after binding to cell membranes or cell membrane-like structure. After DiR labeling, exosomes and MoS\u003csub\u003e2\u003c/sub\u003e@ME were placed in a blank 96-well plate. As shown in \u003cb\u003eFigure S9\u003c/b\u003e, the free DiR is blue-green, while the exosomes are blue after DiR labeling, and there is no obvious change in the appearance of exosomes. The DiR-labelled exosomes (ME-DiR) showed obvious fluorescence, indicating that the exosomes successfully bound to the dye DiR. It should be noted that the DiR-labelled MoS\u003csub\u003e2\u003c/sub\u003e@ME (MoS\u003csub\u003e2\u003c/sub\u003e@ME-DiR) showed weak fluorescence, the same as free dye. The possible reason is that the black MoS\u003csub\u003e2\u003c/sub\u003e shield in MoS\u003csub\u003e2\u003c/sub\u003e@ME absorbs photons/energy from the DiR-labeled membrane structure, thus attenuating the fluorescence received by the instrument detector. Anyway, the above results indicated that the exosomes and MoS\u003csub\u003e2\u003c/sub\u003e@ME were successfully labeled with DiR and could be used for subsequent imaging of living animals.\u003c/p\u003e \u003cp\u003eEach sample labeled with DiR was injected into the tail vein of mice, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Within 48 h, free DiR was mainly distributed in the liver without obvious tumor accumulation, while exosomes and MoS\u003csub\u003e2\u003c/sub\u003e@ME could accumulate at the tumor sites within 2 h, and the accumulation at the tumor sites rose up as time went on, indicating that exosomes and MoS\u003csub\u003e2\u003c/sub\u003e@ME had certain tumor targeting properties and could accumulate in the tumor gradually along with blood circulation. After 48 h, the mice were sacrificed, whose main organs and tumor tissues were taken for \u003cem\u003eex-vivo\u003c/em\u003e imaging. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, free DiR was mainly distributed in the liver and spleen as expected, which meant its clearance by the mononuclear phagocyte system. In addition to the obvious fluorescence observed in the liver and spleen, accumulation of ME and MoS\u003csub\u003e2\u003c/sub\u003e@ME can also be observed in the lung and tumor tissues. Given the nanoscale size of ME and MoS\u003csub\u003e2\u003c/sub\u003e@ME, the possible reason for their accumulation in lung tissue is that the ultrafiltration operation during purification causes the aggregation or shape changes of some particles, resulting in an increase in particle size, which may then be mechanically trapped by pulmonary capillaries. Nonetheless, exosomes and MoS\u003csub\u003e2\u003c/sub\u003e@ME retained accumulation at the tumor sites. On the other hand, the photothermal treatment in this study was triggered by local NIR. It should be difficult for the materials distributed in other parts to produce photothermal effects in the absence of NIR, which reflects the safety advantages of this system. Taken together, exosomes and MoS\u003csub\u003e2\u003c/sub\u003e@ME have certain tumor targeting properties \u003cem\u003ein vivo\u003c/em\u003e and can be used for subsequent drug-targeted therapy. Meanwhile, local irradiation of the tumor site should be performed during subsequent photothermal treatment to reduce the damage to the lungs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e3.3.2 Investigation of the photothermal therapeutic effect of MoS\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e@ME on tumor\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eDuring the photothermal treatment, the temperature changes of tumor sites in mice were monitored. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME had significant effects on elevating the temperature of tumor tissue compared with PBS in the control group. In particular, after the administration of MoS\u003csub\u003e2\u003c/sub\u003e@ME, the lowest temperature of tumor ablation (46\u0026deg;C) could be reached within about 2 min. With the prolongation of NIR irradiation time, the temperature of tumors gradually increased and reached the maximum temperature (52.3\u0026deg;C) at 8 min. Notably, the temperature of the tumor site in the MoS\u003csub\u003e2\u003c/sub\u003e@ME group was far higher than that in the naked MoS\u003csub\u003e2\u003c/sub\u003e group, implying that MoS\u003csub\u003e2\u003c/sub\u003e@ME could increase the accumulation of MoS\u003csub\u003e2\u003c/sub\u003e in the tumor site due to the tumor targeting property of exosomes. These above results indicated that MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME had significant photothermal effects on solid tumors, laying the foundation for subsequent tumor ablation or growth inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter the treatment, the tumor size and the appearance of mice was continuously monitored. It can be seen that no obvious abnormalities were found in other tissues except tumor tissue (\u003cb\u003eFigure S10A\u003c/b\u003e), indicating the safety of the treatment to a certain extent. Within 2 days, the tumor tissues in the MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME groups became black, indirectly reflecting the significant photothermal killing effect of MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME.\u003c/p\u003e \u003cp\u003eWith the time passed by, the tumor size increased in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The degree of increase from large to small was PBS, PBS\u0026thinsp;+\u0026thinsp;NIR group, MoS\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group, and MoS\u003csub\u003e2\u003c/sub\u003e@ME\u0026thinsp;+\u0026thinsp;NIR group. After 20 days, the tumor-bearing mice were sacrificed, and the tumor tissues were peeled off. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, compared with other groups, the tumor volume of the MoS\u003csub\u003e2\u003c/sub\u003e@ME group was significantly reduced under NIR irradiation, and the tumors even completely disappeared. At the end point, compared with the untreated PBS group, the tumor volume of the MoS\u003csub\u003e2\u003c/sub\u003e@ME group decreased under NIR irradiation with statistical difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting that the photothermal treatment had certain antitumor effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Similar to the results of tumor volume, the photothermal treatment could significantly reduce tumor weight compared to other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), and the tumor growth inhibition rate could reach over 60%. In particular, the MoS\u003csub\u003e2\u003c/sub\u003e@ME group can achieve a tumor growth inhibition rate of 75.92% under NIR irradiation, suggesting that MoS\u003csub\u003e2\u003c/sub\u003e@ME has a stronger tumor killing effect. In addition, the survival curves (\u003cb\u003eFigure S10C\u003c/b\u003e) showed that there was no significant difference in the survival function of mice in each group, indicating that the photothermal treatment was relatively mild and had no significant impact on the survival and longevity of mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Blood biochemical and histological tests after treatment\u003c/h2\u003e \u003cp\u003eSubsequently, to verify the safety of MoS\u003csub\u003e2\u003c/sub\u003e@ME, blood taken from the eyeballs of tumor-bearing mice after various treatments was collected to measure the serum liver function, kidney function and other biochemical indicators. As shown in \u003cb\u003eFigure S11\u003c/b\u003e, ALT and AST, as sensitive indicators of liver function damage, were in the normal range in each group, suggesting that MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME may only accumulate in the liver without significant impact on its structure and function. Similarly, the BUN and CRE in each group were normal, suggesting that the kidneys of mice after various treatments were healthy without obvious abnormalities. Moreover, the histological results showed that no obvious pathological damage was found in the main organs of tumor-bearing mice after various treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These results further indicated that MoS\u003csub\u003e2\u003c/sub\u003e@ME had good biological safety. In addition, H\u0026amp;E staining was performed on the tumor tissues of mice after various treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). It was found that compared with other groups, the tumor cells in MoS\u003csub\u003e2\u003c/sub\u003e@ME\u0026thinsp;+\u0026thinsp;NIR group were loosely arranged, the number of cells was significantly reduced, and the staining of the cytoplasm became lighter, suggesting the apoptosis and necrosis of tumor tissues, which confirmed the strong killing effect of MoS\u003csub\u003e2\u003c/sub\u003e@ME on solid tumors under NIR irradiation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4 \u003cem\u003eIn vivo\u003c/em\u003e biosafety evaluation\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e safety of MoS\u003csub\u003e2\u003c/sub\u003e@ME was further investigated by monitoring the body weight of normal mice after tail vein administration and the appearance of major organs after one week (\u003cb\u003eFigures S12 and S13\u003c/b\u003e). As shown in \u003cb\u003eFigure S12A\u003c/b\u003e, there was no significant change in the shape of each organ except that the liver was black in MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME groups, likely from the color of MoS\u003csub\u003e2\u003c/sub\u003e. Within 7 days of administration of MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME, the mice in each group survived with good health: smooth white fur, normal appetite, vigorous activities, and no significant change in body weight (\u003cb\u003eFigure S12B\u003c/b\u003e). H\u0026amp;E staining (\u003cb\u003eFigure S13\u003c/b\u003e) showed that no obvious pathological changes were found in the organs. The results of biosafety evaluation in normal mice were consistent with those of tumor-bearing mice as expected (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), indicating the excellent \u003cem\u003ein vivo\u003c/em\u003e safety of MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSome nano-sized materials are easily cleared by the mononuclear phagocyte system (Mononuclear Phagocyte System, MPS). The current emerging drug delivery system-exosomes, as biological endogenous nanovesicles, are expected to avoid the aforementioned problems[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Studies have demonstrated the incorporation of exosomes and nanomaterials for biological applications such as wound healing[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Exosomes contain proteins such as CD63 and CD81, which are general biomarkers for biological characterization[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The micro structure, size distribution and concentration are important indicators for verifying the quality of exosome isolation[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The results of TEM and DLS showed that the MCF-7 exosomes extracted based on the polyethylene glycol precipitation method had a typical \"cup-disc shape\" with a size distribution of 101\u0026ndash;105 nm, which was in line with the size range of 30\u0026ndash;150 nm for exosomes. Western blotting and flow cytometry showed the MCF-7 exosomes were rich in tetraspanins CD63 and CD81.\u003c/p\u003e \u003cp\u003ePTAs are a key component of photothermal therapy, which convert light energy into thermal effect, causing local protein denaturation and cell membrane damage in tumor tissue, and then inducing tumor cell apoptosis or necrosis[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In PTT, the heat generated by the PTAs is critical and directly determines the tumor therapeutic effect[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The 2D photothermal agent MoS\u003csub\u003e2\u003c/sub\u003e demonstrates attractive photothermal conversion efficiency[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. When activated by NIR, the heating effect of MoS\u003csub\u003e2\u003c/sub\u003e is proportional to the concentration, laser power, and time; at a lower concentration of 30 ppm and a lower power of 0.4 W/cm\u003csup\u003e2\u003c/sup\u003e, the temperature rise of MoS\u003csub\u003e2\u003c/sub\u003e within 5 min is still greater than 32\u0026deg;C, which is obviously higher than that of the control group. At the same time, after 5 \"on-off\" cycles, MoS\u003csub\u003e2\u003c/sub\u003e still maintains similar heating and cooling rates (photothermal conversion efficiency). The above properties of MoS\u003csub\u003e2\u003c/sub\u003e potentially reduce the possibility of repeated drug administration \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eHowever, the lack of targeting and potential safety issue limits the \u003cem\u003ein vivo\u003c/em\u003e application of MoS\u003csub\u003e2\u003c/sub\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this regard, this study plans to employ endogenous exosomes as carriers to construct MoS\u003csub\u003e2\u003c/sub\u003e@ME complex, aiming to endowing targeting capacity. In view of the fact that the size, uniformity of distribution and surface properties of the preparation directly affect its distribution and metabolism in body tissues[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], this study conducted a series of investigations on the basic properties of MoS\u003csub\u003e2\u003c/sub\u003e@ME, including morphology, particle size, photothermal properties and protein ingredients. The consistence of MoS\u003csub\u003e2\u003c/sub\u003e@ME and MoS\u003csub\u003e2\u003c/sub\u003e on heating rate and heating and cooling cycle curves suggests that MoS\u003csub\u003e2\u003c/sub\u003e@ME retained the photothermal properties of MoS\u003csub\u003e2\u003c/sub\u003e. The MoS\u003csub\u003e2\u003c/sub\u003e@ME displayed concentrated particle size distribution (ranging from 85 to 112 nm) and retained protein components. The above results show that the preparation for MoS\u003csub\u003e2\u003c/sub\u003e delivery is successfully prepared.\u003c/p\u003e \u003cp\u003eMoS\u003csub\u003e2\u003c/sub\u003e@ME involves two important elements of MoS\u003csub\u003e2\u003c/sub\u003e and exosomes in our design. Among them, since molybdenum is one of the essential trace elements for animals and humans[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], MoS\u003csub\u003e2\u003c/sub\u003e has high biocompatibility in 2D transition metal sulfide-based PTAs[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The exosomes naturally have good biocompatibility due to their cell membrane-like structures[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The toxicity of the above materials and their preparation were analyzed by CCK-8 and live/dead staining. The results showed that none of the above materials and preparation obviously affected cell survival in tumor cells or normal cell lines (cell viability\u0026thinsp;\u0026gt;\u0026thinsp;80%, both in human and murine cells) in the absence of NIR, and the preparation did not cause hemolysis in red blood cells, indicating that the preparation retains the biocompatibility of the raw material and has considerable \u003cem\u003ein vitro\u003c/em\u003e safety. At the same time, under NIR triggering, the preparation showed near 100% killing of MCF-7 and 4T1 cells, indicating that it has excellent light-triggered photothermal conversion ability and tumor killing effect. The above discovery had provided importation indication for the subsequent \u003cem\u003ein vivo\u003c/em\u003e animal experiments.\u003c/p\u003e \u003cp\u003eIn addition, the exosomes used in the system theoretically have homologous targeting and can actively target tumor cells. Because the biogenesis of exosomes determines that they naturally carry a large number of donor cell information and cell type-specific proteins found in the parent cell membrane, the adhesion proteins on their surface have been shown to be parental cell-prone and can attach to target cells[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Qualitative and quantitative analysis by CLSM and flow cytometry found that the uptake of exosomes was related to incubation time and cell type. The cellular uptake of exosomes within 6 h was proportional to incubation time, and was basically saturated at 4 h. Compared with normal MCF-10A cells, tumor exosomes have higher cellular uptake in tumor MCF-7 and 4T1 cells; flow cytometry results show that the positive rate difference between MCF-7 and MCF-10A uptake can reach 60%. After incorporation of MoS\u003csub\u003e2\u003c/sub\u003e, this difference decreased, but still maintained a higher uptake by MCF-7 cells. The above results are basically in line with expectations, which preliminarily indicate that the preparation presents tumor cell selectivity.\u003c/p\u003e \u003cp\u003e4T1 subcutaneous xenograft tumor model was established for verification of \u003cem\u003ein vivo\u003c/em\u003e biodistribution and photothermal therapy effects with Balb/c mice. The growth and metastasis characteristics of 4T1 cells are very similar to breast cancer in humans, and thus they are widely used as a model of human breast cancer[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. With DiR labelling, the tissue distribution of the preparation in tumor-bearing mice was investigated. Mouse tail vein administration of DiR was used to rule out the possibility of accumulation of the dye itself in the tumor. However, semi-quantitative fluorescence showed that the mouse tumor site showed weaker fluorescence after administration of DiR than before administration. The possible reason is that the dye itself is prone to aggregation. In order to completely rule out the aggregation of DiR dye and its possible impact on the kinetics of the preparation, future experiments may consider directly modifying fluorescent molecules on exosomes/hybrid vesicles, or use emerging technologies such as photoacoustic imaging to eliminate their interference[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. \u003cem\u003eIn vivo\u003c/em\u003e biodistribution results showed that as the carrier or preparation circulates in the blood, its fluorescence at the tumor site gradually increases, and the results are significantly different from the control group, indicating that the preparation has certain tumor targeting properties. However, compared with other major metabolic organs such as liver and spleen, the targeting ability is still not excellent. This can be explained by the results from some reports: when injected intravenously, some unmodified tumor-derived exosomes can be rapidly taken up by MPS in the liver and spleen[\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In particular, some studies have shown that exosomes have higher tumor cell uptake than dye when injected intratumorally, suggesting that the targeting of exosomes is more likely to be reflected in the exchange of information with neighboring cells[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In view of this, in the future, modification of exosomes targeting ligands or intratumorally local injection can be considered to highlight their active targeting.\u003c/p\u003e \u003cp\u003eIn PTT, the light source is the \"switch\" that triggers its therapeutic effect, usually visible light (400\u0026ndash;800 nm) or near-infrared light (NIR, 800\u0026ndash;1700 nm). Compared with visible light, which can be absorbed by many endogenous substances in biological tissues and cause scattering[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], NIR's longer wavelength can reduce tissue scattering and increase penetration depth[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], so it is considered to be favored. However, current PTAs are mostly concentrated in the NIR region (NIR-I, 800\u0026ndash;1000 nm), which still has limited penetration depth and heat resistance caused by thermal shock[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Taking this into consideration, our work employed 1064 nm laser as irradiation light source, which is in the second zone near-infrared light (NIR-II, 1000\u0026ndash;1700 nm) and can achieve lower tissue scattering and deeper penetration into the skin tissue, thereby realizing improved therapeutic efficacy [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. At the same time, in view of the complexity of the environment and metabolic processes of cells in animals compared with those \u003cem\u003ein vitro\u003c/em\u003e, a power density of 0.48 W/cm\u003csup\u003e2\u003c/sup\u003e, which was slightly higher than that in cell experiments (0.4 W/cm\u003csup\u003e2\u003c/sup\u003e), was finally selected for animal experiments and the laser irradiation time was extended. At 2 min, the temperature of tumor site treated with MoS\u003csub\u003e2\u003c/sub\u003e@ME has increased by more than 18℃, which was similar to the results of \u003cem\u003ein vitro\u003c/em\u003e experiment, suggesting that the system has strong tissue penetration in a short time. This photothermal condition is still relatively mild, and no obvious skin damage was seen on tumor-bearing mice without PTAs, indicating that NIR-II at this power has good safety in normal tissues. When MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME were administered, black scabs appeared at the tumor site of mice, reflecting the powerful lethality of the photothermal treatment. It has been reported that when tissue temperature rises to 42\u0026deg;C, irreversible tissue damage occurs; when it rises to 42\u0026ndash;46\u0026deg;C, cell necrosis will occur within 10 minutes; and when the temperature reaches 46\u0026ndash;52\u0026deg;C, cells will form microvascular thrombosis, local ischemia leads to rapid death[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. It can be seen from the thermal imaging and heating curve of animal tumor sites that after treatment with MoS\u003csub\u003e2\u003c/sub\u003e and MoS\u003csub\u003e2\u003c/sub\u003e@ME, the temperature of tumor site can reach 46\u0026deg;C in a short period of time. It is worth noting that MoS\u003csub\u003e2\u003c/sub\u003e@ME group reaches the killing temperature faster than the MoS\u003csub\u003e2\u003c/sub\u003e group. The final tumor inhibition rate also confirmed this judgment. The MoS\u003csub\u003e2\u003c/sub\u003e@ME exhibited stronger inhibition of tumor growth than MoS\u003csub\u003e2\u003c/sub\u003e alone during the whole treatment periods. The results suggested that the strategy was simple but potent, which can achieve tumor photothermal ablation at lower laser power and in a shorter treatment course. For example, our preparation needed only single treatment with 0.48 W/cm\u003csup\u003e2\u003c/sup\u003e, while the MoS\u003csub\u003e2\u003c/sub\u003e coated with hyaluronic acid (HA) required 1 W/cm\u003csup\u003e2\u003c/sup\u003e and triple treatment[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In another case, MoS\u003csub\u003e2\u003c/sub\u003e modified with polyethylene glycol (PEG) and vanadium (V- MoS\u003csub\u003e2\u003c/sub\u003e@PEG) employed 808 nm laser (NIR-I, relatively week penetration) at 0.6 W/cm\u003csup\u003e2\u003c/sup\u003e for 4-times irradiation[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In addition, the study also examined the safety of the treatment from the perspectives of normal mice and tumor-bearing mice and their main organs. Body weight, histological staining, blood biochemistry and other indicators all showed that the treatment had no significant impact on the mice's daily activities and the structure and function of important organs. Based on the above, after intravenous injection of MoS\u003csub\u003e2\u003c/sub\u003e@ME for 24 h, the photothermal therapy mode exposing the tumor site to 0.48 W/cm\u003csup\u003e2\u003c/sup\u003e 1064 nm NIR for 10 min has significant tumor killing effect and good safety \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this work, we have successfully prepared MoS\u003csub\u003e2\u003c/sub\u003e@ME for advanced photothermal tumor therapy. With the help of ultrasonic self-assembly membranes, MoS\u003csub\u003e2\u003c/sub\u003e nanodots were incorporated into MCF-7 exosomes. The obtained MoS\u003csub\u003e2\u003c/sub\u003e@ME efficiently killed MCF-7 and 4T1 cells under NIR irradiation. In the absence of NIR, it did not show toxicity to tumor cells MCF-7, 4T1 and normal cells MCF-10A, L929, indicating its good biocompatibility and \u003cem\u003ein vitro\u003c/em\u003e safety. Moreover, MoS\u003csub\u003e2\u003c/sub\u003e@ME basically retained the tumor cell selectivity of exosomes and could achieve \u003cem\u003ein vivo\u003c/em\u003e tumor targeting. Furthermore, it presented significant tumor tissue heating effects in 4T1 tumor-bearing mice as soon as the NIR was switched. Despite mild laser conditions, it showed significantly lower tumor volume and weight, and higher tumor growth inhibition rate than naked MoS\u003csub\u003e2\u003c/sub\u003e. In a word, the results of mice study, including tumor appearance, volume and weight, survival rate, body weight and major organ changes, showed that the delivery system based on MoS\u003csub\u003e2\u003c/sub\u003e and exosomes could achieve \u003cem\u003ein vivo\u003c/em\u003e tumor targeting, efficient photothermal anti-tumor and certain biological safety. Generally, a new strategy of constructing biomaterials for PTT is demonstrated in this paper, which may provide a new insight for the development of PTT.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics and consent to participate declarations\u003c/h2\u003e \u003cp\u003eAll the animal experimental protocols were approved by the Animal Research Committee of Guangdong Medical University.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work is supported by Guangdong Basic and Applied Basic Research Foundation (2021B1515140006), Special projects in key areas for general colleges and universities of Guangdong Province (2021ZDZX2061, 2024ZDZX2068), Featured Innovation Projects for General Colleges and Universities of Guangdong Province (2022KTSCX042), Medical Scientific Research Foundation of Guangdong Province (A2023241), Funds for PhD Researchers of Guangdong Medical University in 2023 (4SG23184G and 4SG23233G), Dongguan Social Development Technology Project-Key Project (20231800940842), Songshan Lake Science and Technology Correspondent Project (20234403-01KCJ-G), Construction Project of Nano Technology and Application Engineering Research Center of Guangdong Medical University (4SG24179G), College Students\u0026rsquo; innovation and entrepreneurship training program (202410571010, S202410571059, S202410571061, S202410571054, 202310571001, 202310571004, 202310571024), College Students\u0026rsquo; innovation experiment program of Guangdong Medical University (FYDM004), and Discipline Construction Project of Guangdong Medical University (1019K20220003).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLiyan Wang: Writing\u0026ndash;original draft, Methodology, Formal analysis, Data curation, Conceptualization. Huizhi Chen: Writing\u0026ndash;original draft, Methodology, Formal analysis, Data curation. Haiyan Qiu: Investigation, Formal analysis, Data curation. Zhenyu Xie: Validation, Supervision. Shah Zada: Investigation, Formal analysis, Data curation. Jianbo Sun: Validation, Supervision. Chengyu Lu: Writing\u0026ndash;review \u0026amp; editing, Resources, Project administration, Funding acquisition. Zhan Zhou: Writing\u0026ndash;review \u0026amp; editing, Resources, Project administration, Funding acquisition. Xinsheng Peng: Funding acquisition, Project administration, Resources, Writing\u0026ndash;review \u0026amp; editing. Ruizheng Liang: Project administration, Resources, Writing\u0026ndash;review \u0026amp; editing. Yubin Zhou: Funding acquisition, Project administration, Resources, Writing\u0026ndash;review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJung HS, Verwilst P, Sharma A, Shin J, Sessler JL, Kim JS: \u003cstrong\u003eOrganic molecule-based photothermal agents: an expanding photothermal therapy universe.\u003c/strong\u003e \u003cem\u003eChem Soc Rev \u003c/em\u003e2018, \u003cstrong\u003e47:\u003c/strong\u003e2280-2297.\u003c/li\u003e\n\u003cli\u003eZhou Z, Wang X, Zhang H, Huang H, Sun L, Ma L, Du Y, Pei C, Zhang Q, Li H, et al: \u003cstrong\u003eActivating Layered Metal Oxide Nanomaterials via Structural Engineering as Biodegradable Nanoagents for 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Biotechnology \u003c/em\u003e2022, \u003cstrong\u003e10\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eKenry, Duan Y, Liu B: \u003cstrong\u003eRecent Advances of Optical Imaging in the Second Near-Infrared Window.\u003c/strong\u003e \u003cem\u003eAdv Mater \u003c/em\u003e2018, \u003cstrong\u003e30:\u003c/strong\u003ee1802394.\u003c/li\u003e\n\u003cli\u003eLi X, Lovell JF, Yoon J, Chen X: \u003cstrong\u003eClinical development and potential of photothermal and photodynamic therapies for cancer.\u003c/strong\u003e \u003cem\u003eNat Rev Clin Oncol \u003c/em\u003e2020, \u003cstrong\u003e17:\u003c/strong\u003e657-674.\u003c/li\u003e\n\u003cli\u003eYe H, Yan J, Ge C, Wu F, Zhu J, Yin M, Xie L, Zhou Z, Yin L: \u003cstrong\u003eTumoral/exosomal PD-L1 silencing reinforces mild photothermal therapy by relieving systemic and local immunosuppression.\u003c/strong\u003e \u003cem\u003eChemical Engineering Journal \u003c/em\u003e2024, \u003cstrong\u003e483:\u003c/strong\u003e149093.\u003c/li\u003e\n\u003cli\u003eWang H, Xia P, Kurboniyon MS, Fang S, Huang K, Ning S, Jin G, Zhang L, Wang C: \u003cstrong\u003eV-doped MoS\u003csub\u003e2\u003c/sub\u003e nanozymes providing reactive oxygen species and depleting glutathione for photothermally-enhanced nanocatalytic therapy.\u003c/strong\u003e \u003cem\u003eFrontiers in Pharmacology \u003c/em\u003e2024, \u003cstrong\u003e15\u003c/strong\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Photothermal therapy, Targeting, Exosomes, MoS2","lastPublishedDoi":"10.21203/rs.3.rs-6226702/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6226702/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhotothermal therapy (PTT) has been an attractive tumor treatment strategy in recent years. Two-dimensional molybdenum disulfide (MoS\u003csub\u003e2\u003c/sub\u003e)-based nanomaterials with high photothermal efficiency is a critical candidate for PTT. However, the tumor-targeting capability extremely needs to be improved for effective tumor treatment. In this work, we combine the MoS\u003csub\u003e2\u003c/sub\u003e nanodots with exosomes, native vesicles secreted from living cells, to construct a novel exosomes/MoS\u003csub\u003e2\u003c/sub\u003e complex (MoS\u003csub\u003e2\u003c/sub\u003e@ME) for effective tumor-targeted PTT. Through ultrasonic self-assembly membranes, MoS\u003csub\u003e2\u003c/sub\u003e nanodots are incorporated into MCF-7 exosomes. Similar to free MoS\u003csub\u003e2\u003c/sub\u003e, MoS\u003csub\u003e2\u003c/sub\u003e@ME shows significant photothermal conversion effect, causing nearly 100% necrosis proportion of MCF-7 and 4T1 cells under 1064 nm laser irradiation within 5 min (0.4 W/cm\u003csup\u003e2\u003c/sup\u003e) \u003cem\u003ein vitro\u003c/em\u003e. In particular, MoS\u003csub\u003e2\u003c/sub\u003e@ME presents noteworthy affinity for tumor cells, and \u003cem\u003ein vivo\u003c/em\u003e studies further prove that it could accumulate at the tumor site efficiently. After intravenous injection with MoS\u003csub\u003e2\u003c/sub\u003e@ME plus NIR irradiation, the temperature of tumor site in 4T1 tumor-bearing mice could reach 46\u0026deg;C within a short time (~\u0026thinsp;2 min). Notably, with the prolongation of NIR irradiation time, the temperature of tumors gradually increases and reaches the maximum temperature (52.3\u0026deg;C) at 8 min, which is far higher than that in the free MoS\u003csub\u003e2\u003c/sub\u003e group. More importantly, PTT using MoS\u003csub\u003e2\u003c/sub\u003e@ME exhibits much more effective antitumor therapy, as the tumor volume and tumor weight of mice in the MoS\u003csub\u003e2\u003c/sub\u003e@ME group are significantly lower than those in the PBS and MoS\u003csub\u003e2\u003c/sub\u003e groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and even the tumor disappears completely. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies demonstrate that the MoS\u003csub\u003e2\u003c/sub\u003e@ME shows excellent targeting capacity and photothermal effect, achieving effective photothermal cancer therapy. This work is expected to overcome the shortcomings of some photothermal materials, aiming to improve safety and effectiveness. The exosome-incorporated design strategy may pave a new way for molybdenum-based tumor-targeted PTT.\u003c/p\u003e","manuscriptTitle":"Exosomes/MoS2 complex for targeting and effective photothermal therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 09:54:00","doi":"10.21203/rs.3.rs-6226702/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-24T03:24:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-31T18:39:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-29T09:39:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-29T06:32:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-25T03:20:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"10128648178772527525966491495137803874","date":"2025-03-22T04:57:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55667300962946422168141189184757294157","date":"2025-03-22T01:23:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196279038535474851659594456796356275573","date":"2025-03-21T21:07:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180070569359708866424391449919808288837","date":"2025-03-21T19:35:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-21T16:13:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-17T04:19:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-17T04:18:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-03-14T13:13:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a834b9cd-de1b-462f-9a06-1074c4ed752f","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:00:01+00:00","versionOfRecord":{"articleIdentity":"rs-6226702","link":"https://doi.org/10.1186/s12951-025-03665-8","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2025-09-29 15:57:08","publishedOnDateReadable":"September 29th, 2025"},"versionCreatedAt":"2025-03-31 09:54:00","video":"","vorDoi":"10.1186/s12951-025-03665-8","vorDoiUrl":"https://doi.org/10.1186/s12951-025-03665-8","workflowStages":[]},"version":"v1","identity":"rs-6226702","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6226702","identity":"rs-6226702","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}