Maximizing Oxaliplatin's Impact on EGFR+ Colorectal Cancer Through Targeted Extracellular Vesicles

Maximizing Oxaliplatin's Impact on EGFR+ Colorectal Cancer Through Targeted Extracellular Vesicles | 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 Maximizing Oxaliplatin's Impact on EGFR+ Colorectal Cancer Through Targeted Extracellular Vesicles Shang-Tao Chien, Yi-Jung Huang, Ming-Yii Huang, Yi-Ping Fang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4291698/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Aug, 2024 Read the published version in Cancer Nanotechnology → Version 1 posted 11 You are reading this latest preprint version Abstract Purpose: To investigate the ability of extracellular vesicles (EVs) to deliver oxaliplatin to epidermal growth factor receptor (EGFR + ) colorectal cancer cells and increase oxaliplatin’s cytotoxicity. Method: Oxaliplatin was passively loaded into a stable cell line expressing cetuximab in membranes. EVs were collected and characterized for size, and their ability to target EGFR + cells was tested. Cytotoxicity experiments were performed, and a xenograft cancer animal model was used to confirm the specific accumulation of oxaliplatin-loaded EVs with cetuximab-expressing membranes in EGFR + cells. Results: EVs with cetuximab-expressing membranes were successfully produced and used to encapsulate oxaliplatin, resulting in consistently sized oxaliplatin-loaded EVs with cetuximab-expressing membranes. The oxaliplatin-loaded EVs with cetuximab-expressing membranes were specifically internalized by EGFR + cells, leading to significant cytotoxic effects on these cells. In the animal model, the oxaliplatin-loaded EVs with cetuximab-expressing membranes accumulated specifically in EGFR + cells and significantly enhanced oxaliplatin’s therapeutic efficacy against EGFR + cancer cells. Conclusion: EVs with membrane-expressed bioactive molecules are a promising strategy for delivering therapeutic agents to EGFR + colorectal cancer cells. Extracellular Vesicles Oxaliplatin Delivery and EGFR+ Colorectal Cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights EVs with cetuximab-expressing membranes were used to encapsulate oxaliplatin and target EGFR + cells. Oxaliplatin-loaded EVs with cetuximab-expressing membranes exhibited significant cytotoxic effects on EGFR + cells. In a xenograft cancer animal model, oxaliplatin-loaded EVs with cetuximab-expressing membranes accumulated specifically in EGFR + cells and significantly enhanced oxaliplatin’s therapeutic efficacy against EGFR+ cancer cells. EVs with targeted membrane expression of drugs are a promising strategy for delivering therapeutic agents to specific cell populations. Introduction Extracellular vesicles (EVs) are secreted by the cells themselves, which makes them biocompatible. Compared to synthetic drug carriers, EVs undergo more natural excretion and metabolism processes in the body. In addition, EVs can reduce interactions with the immune system, thereby mitigating potential immune reactions. These characteristics make them excellent carriers for the delivery of therapeutic drugs [1–4]. For example, Somiya et al. demonstrated excellent biocompatibility of EVs in vivo, as they were cleared without adverse reactions. The toxicity, immunology, and immunogenicity of EVs have also been evaluated, with results showing acceptable biocompatibility in vivo [5]. Wu et al. highlighted that EVs, being products of endogenous secretion, exhibit good biocompatibility within the body and can reduce interactions with the immune system. These attributes confer potential advantages to EVs as drug delivery systems and mitigate the risk of immune reactions [6]. Targeting the specificity of EVs is crucial for their application in disease treatment. For instance, Zhu et al. demonstrated that conjugated Hypo-Exo with an ischemic myocardium-targeted peptide facilitates ischemic cardiac repair by ameliorating cardiomyocyte apoptosis. This indicates that particular peptides can enable EVs to acquire targeted specificity [7]. In breast cancer treatment, the targeting of EVs using anti-human CD3 and anti-human HER2 antibodies has been employed to generate SMART-Exos that dual-target T cells to CD3 and HER2 receptors associated with breast cancer. This approach enhances the efficacy of targeting of HER2-positive breast cancer cells by T cells [8–13]. In lung cancer treatment, ligands that bind to the epidermal growth factor receptor (EGFR) can be modified to attach to the surface of EVs to increase their binding and uptake by EGFR-positive tumor cells. In previous studies, EVs have been co-incubated with epidermal growth factor or EGFR antibodies to allow the ligands to bind to the EV surface and the targeted effects of the modified EVs on tumor cells have been evaluated using cell-based assays and animal models [14–19]. Although targeted EVs have been applied in breast cancer and lung cancer, their application in colorectal cancer remains unclear. In this study, we passively loaded oxaliplatin into a stable cell line expressing cetuximab, an EGFR inhibitor, in the membrane and collected its EVs to confirm their ability to load oxaliplatin and target EGFR to increase the cytotoxicity of oxaliplatin in EGFR + colorectal cancer cells (Fig. 1). We constructed EVs able to express cetuximab on the membrane and then confirmed cetuximab expression on the EV membrane. We also confirmed whether the EVs changed their particle size after encapsulating oxaliplatin. We investigated whether EVs with cetuximab-expressing membranes could be internalized by EGFR + colorectal cancer cells to increase cytotoxicity. Finally, we established a xenograft colorectal animal model to confirm the tumor-specific accumulation of oxaliplatin-encapsulating EVs with cetuximab-expressing membranes and their therapeutic effect on EGFR + colorectal cancer. Results Extracellular vesicles with cetuximab-expressing membranes were successfully constructed To enable cetuximab expression on the membranes of EVs, we constructed stable HEK cell lines with their membranes expressing the cetuximab antibody. We cultured HEK293 cells and HEK293 cells secreting EVs with cetuximab-expressing membranes. We then collected the cell supernatant and subjected it to differential centrifugation to isolate HEK293 EVs and HEK293 EVs with cetuximab-expressing membranes. Western blot analysis was performed to confirm the expression of cetuximab in HEK293 cells, in HEK293 cells with cetuximab-expressing membranes, in HEK293 EVs, and in HEK293 EVs with cetuximab-expressing membranes, as well as to assess the accuracy of our sample collection. In addition, human Fab analysis was performed to confirm successful cetuximab expression. We also analyzed specific markers of EVs, such as Hsp70, CD81, and CD9. The results indicated the presence of their respective specific molecules in EVs compared to the cell groups, demonstrating successful collection of EVs from both HEK293 cells and HEK293 cells with cetuximab-expressing membranes. Further comparison of the expression levels of cetuximab in the EVs of HEK293 cells and the EVs of HEK293 cells with cetuximab-expressing membranes showed that both the cells and their EVs expressed the human antibody, indicating successful establishment of the HEK293 cell line with cetuximab-expressing membranes and EVs with cetuximab-expressing membranes (Fig. 2). Extracellular vesicles with cetuximab-expressing membranes were successfully loaded with oxaliplatin To confirm the successful encapsulation of oxaliplatin by EVs with cetuximab-expressing membranes, we introduced oxaliplatin into one group of cells secreting EVs with membrane-expressed cetuximab, while another group of similar cells was not treated with oxaliplatin. UV irradiation was then applied for 30 min, followed by the collection of EVs. After subjecting the collected EVs to high-speed centrifugation, we analyzed them using transmission electron microscopy (Fig. 3A) and nanoparticle tracking (Fig. 3B). The results of transmission electron microscopy showed that the particles of oxaliplatin-encapsulating EVs with cetuximab-expressing membranes were slightly larger than similar vesicles without oxaliplatin encapsulation (152.8 ± 1.4 nm vs. 142.4 ± 1.2 nm). However, the nanoparticle tracking analysis did not find a significant difference in particle size. These results confirm the successful establishment of oxaliplatin-encapsulating EVs with cetuximab-expressing membranes. Extracellular vesicles with cetuximab-expressing membranes can undergo EGFR + colorectal cancer-specific internalization To confirm whether EVs with cetuximab-expressing membranes can undergo EGFR+ colorectal cancer-specific internalization, we coated rhodamine B on EVs with normal membranes and those with membranes expressing cetuximab. These vesicles were separately added to HCT116 (EGFR + ) and SW620 (EGFR - ) cells and incubated for a specific period. Afterward, the vesicles that did not bind or were not internalized into the cells were washed away with PBS. The results showed significant internalization in the HCT116 (EGFR + ) group compared to the SW620 (EGFR - ) group (Fig. 4A vs. Fig. 4B). In addition, in the comparison of the cell-derived vesicles with membranes able to express cetuximab and those without in the HCT116 group, the EVs with cetuximab-expressing membranes exhibited more pronounced internalization (Fig. 4A). Oxaliplatin-encapsulating extracellular vesicles with cetuximab-expressing membranes increase the cytotoxicity of oxaliplatin against colorectal cancer To confirm whether oxaliplatin-encapsulating EVs with cetuximab-expressing membranes can enhance the cytotoxicity of oxaliplatin against EGFR + colorectal cancer cells, we separately cultured HCT116 and SW620 cells and treated them with EVs encapsulating oxaliplatin, or EVs with cetuximab-expressing membranes, or oxaliplatin-encapsulating EVs with cetuximab-expressing membranes. After 1 hour, the cells were washed with PBS and incubated in fresh culture medium for 48 hours. Cell viability was then analyzed. The results showed that compared to oxaliplatin-encapsulating EVs with normal membranes, oxaliplatin-encapsulating EVs with cetuximab-expressing membranes exhibited higher cytotoxicity in EGFR + cells (Fig. 5). However, this phenomenon was not observed in EGFR - cells. Based on these findings, we suggest that oxaliplatin-encapsulating EVs with cetuximab-expressing membranes can enhance the killing effect of oxaliplatin specifically on EGFR + cancer cells. The specificity of accumulation of rhodamine B-loaded extracellular vesicles with cetuximab-expressing membranes in cancer cells in a xenograft model We performed an additional experiment to confirm the specific accumulation of EVs with membrane-expressed cetuximab in tumor cells. We injected SW620 cells into the left leg and HCT116 cells into the right leg of mice, allowing them to grow into a tumor sized 50 mm 3 . Then, we administered rhodamine B-loaded EVs with normal membranes and rhodamine B-loaded EVs with cetuximab-expressing membranes into the tail vein. We determined the absorbance of a 580-nm wavelength at 0 min, 30 min, 120 min, 180 min, 240 min, and 360 min. The results indicated that compared to the group treated with EVs with normal membranes, the group treated with EVs with cetuximab-expressing membranes showed significant accumulation of rhodamine B in the tumor. Furthermore, compared to the fluorescence accumulation in SW620 cells (Fig. 6A), the fluorescence accumulation in HCT116 cells was higher after 120 min (Fig. 6B). This phenomenon suggests that membrane-expressed cetuximab can selectively deliver compounds to target tumors. The therapeutic efficacy of oxaliplatin against EGFR + colorectal cancer is enhanced when it is encapsulated in extracellular vesicles with cetuximab-expressing membranes To assess the treatment efficacy against EGFR + colorectal cancer of oxaliplatin encapsulated in EVs with cetuximab-expressing membranes, we subcutaneously injected SW620 and HCT116 cells into the left legs of mice. Once the tumors reached a volume of 50 mm 3 , we administered PBS or 5 mg/kg/3 days (cumulative 3 doses) of oxaliplatin, EVs with cetuximab-expressing membranes, or oxaliplatin-encapsulating EVs with cetuximaexpressing membranes. Tumor volume was measured every three days until day 30. In the HCT116 tumor model, the results showed that the tumor volume was smaller in the group treated with EVs with cetuximab-expressing membranes compared to the PBS group, and the tumor volume was even smaller in the group treated with oxaliplatin-encapsulating EVs with cetuximab-expressing membranes compared to the oxaliplatin group (Fig. 7A). However, no significant differences were observed in the SW620 tumor model (Fig. 7B). These results indicate that oxaliplatin-encapsulating EVs with cetuximab-expressing membranes had the highest efficacy against HCT116 tumor cells (Fig. 7A). Materials And Methods Cell lines and cell culture HEK293, HCT116 (EGFR + ), and SW620 (EGFR - ) cell lines (Bioresource Collection and Research Center, Hsinchu 300193, Taiwan) were cultured in Dulbecco's Modified Eagle Medium (Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% bovine calf serum (Cytiva, Washington, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific Inc., Waltham, MA, USA). The cells were maintained in an incubator with 5% CO 2 at 37 °C. Establishment of extracellular vesicles with membrane-expressed cetuximab and oxaliplatin encapsulation The light and heavy chains of cetuximab (NCBI, US) fused to the immunoglobulin C2-type extracellular transmembrane–cytosolic domains of the mouse B7-1 antigen (eB7) [20] were cloned into the pLKO_AS2 vector (National Core Facility for Biopharmaceuticals, Taipei, Taiwan) using NheI, AscI, BglII, and BstxI restriction enzymes. The plasmid was then transformed into TOP 10 cells (Thermo Fisher Scientific Inc.) for amplification and extraction. The lentiviral vector was produced by co-transfecting pMD.G, pCMVΔR8.91 (National Core Facility for Biopharmaceuticals), and pLKO_AS2 cetuximab into HEK293 cells. Stable HEK293 cell lines that can secrete EVs with cetuximab-expressing membranes were selected using 1 mg/ml of neomycin sulfate (Thermo Fisher Scientific Inc.) after infection with virus. The HEK293 cells were cultured in serum-free medium and treated with 5mg/ml of oxaliplatin (Merck, Darmstadt, Germany) and irradiated with UVB once at a dose of 30 mJ/cm 2 for 24 hours The EVs with cetuximab-expressing membranes were collected from the cells. Collection of extracellular vesicles with cetuximab-expressing membranes and Western blot analysis Collecting EVs from drug-treated or untreated cell lines involved a series of centrifugation steps. The conditioned medium from the cells was first centrifuged at 300 × g for 10 min at 4 °C to remove cellular debris. The supernatant was then subjected to further centrifugation at 2000 × g for 10 min at 4 °C to remove larger microvesicles. Next, the supernatant was centrifuged at 10,000 × g for 30 min at 4 °C to pellet smaller EVs. Finally, the pellet was resuspended in physiological saline and subjected to ultracentrifugation at 100,000 × g for 1.5 hours at 4 °C to collect the extracellular vesicle pellet. For Western blot analysis, the collected EVs with cetuximab-expressing membranes, at 10 ug/well, were separated by SDS-PAGE and transferred onto a nitrocellulose NC membrane (Schleicher and Schuell, Einbeck, Germany). The NC membranes were probed with Mouse Anti-Human IgG Fab Antibody [HRP] (GenScript Biotech, Piscataway, NJ, USA), monoclonal HSP70 antibody (3A3, MA3-006; Thermo Fisher Scientific Inc.), anti-CD81 antibody (ab155760; abcam, Cambridge, UK), CD9 (D8O1A) Rabbit mAb #13174 (abcam), peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG, Fc Fragment Specific (115-035-008, 1:1000; Jackson ImmunoResearch, West Grove, PA, USA), or Rabbit Anti-Goat IgG Antibody, HRP conjugate, (rabbit IgG, AP106P, 1:1000; Sigma-Aldrich) and visualized with the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500; Merck, Rahway, NJ, USA), according to the manufacturer’s protocol. Transmission electron microscopy and nanoparticle tracking analysis Two micrograms of EVs were pipetted (5 ml) onto formvar-coated copper grids (FF200-Cu; Electron Microscopy Sciences, Hatfield, PA, USA) and allowed to settle for 20 min at room temperature. Excess phosphate-buffered saline (PBS) was removed by wicking with filter paper before fixation using a 2% paraformaldehyde, 2% glutaraldehyde, and 0.05 M phosphate solution for 2 min. Grids were washed three times with distilled water prior to application of 1% phosphotungstic acid counterstain for 1 min. Excess liquid was removed by wicking with filter paper, and the grids were allowed to dry overnight at room temperature. Grids were analyzed using a transmission electron microscope (Technai G2 20; FEI, Hillsboro, OR, USA). EVs were visualized and quantified using a nanoparticle tracking analyzer (Nanosight NS300; Nanosight Ltd., Amesbury, UK) with 70 mW laser with a wavelength of 405 nm. Urine samples were diluted in PBS at a ratio of 1:100, while serum samples were diluted at 1:1000. Duplicate measurements were recorded for each sample. Rhodamine B packaging by extracellular vesicles with cetuximab-expressing membranes and fluorescent staining After mixing 5 mg/ml of rhodamine B (TCI, Tokyo, Japan) with 2 mg/ml of EVs with cetuximab-expressing membranes, the reaction was allowed to proceed for 1 hour. The mixture was then washed with PBS to separate the free rhodamine B and EVs by centrifugation at 100,000 × g for 10 min at 4 °C in an ultracentrifuge (Optima™ XPN; Beckman Coulter, Brea, California, USA). The pellet was collected and resuspended in PBS for further use. Then, 2 × 10 3 SW620 and HCT116 cells were separately seeded on glass slides and 5 μM of rhodamine B-loaded EVs with cetuximab-expressing membranes was added to each cell culture and incubated for 90 min. The supernatant was removed and cells were washed with PBS. The cells were then fixed with 10% formalin (KINGFEX CO., LTD., Taipei, Taiwan) and stained with CellTracker™ Green CMFDA Dye (Thermo Fisher Scientific Inc.) and DAPI (Merck, Darmstadt, Germany) and mounted with mounting media (Thermo Fisher Scientific Inc.). Fluorescent images were acquired with a confocal fluorescence microscope (FV1000; Olympus, Tokyo, Japan). Cell viability assay Cells were plated in 96-well plates (2000 cells/well) overnight and serially diluted EVs, oxaliplatin-encapsulating EVs, EVs with cetuximab-expressing membranes, or oxaliplatin-encapsulating EVs with cetuximab-expressing membranes were added.Cell viability was detected using the ATPlite kit (510-17281; PerkinElmer, Chennai, India) and the luminesce value was measured using a multimode plate reader (VICTORTM X2; PerkinElmer). Establishment of an ectopic cancer mouse model and analysis of the accumulation of rhodamine B-loaded extracellular vesicles with cetuximab-expressing membranes Eight-week-old male nude mice (BALB/cAnN.Cg-Foxn1nu/CrlNarl) obtained from the National Laboratory Animal Center, Taiwan, were used in this study. The HCT116 and SW620 cell suspensions (2 × 10 6 in PBS) were subcutaneously inoculated into the right hind leg of the mice. Tumors were allowed to grow until they reached approximately 50 mm 3 in size. Rhodamine B-loaded EVs were injected into the tail vein at a dose of 5 mg/kg, and the accumulation of tumor fluorescence was measured at different time points using IVIS (PerkinElmer, Inc., Waltham, MA, USA). Oxaliplatin-encapsulating extracellular vesicles with cetuximab-expressing membranes enhance the therapeutic effect of oxaliplatin on EGFR+ cancer cells When the tumors reached approximately 50 mm 3 in size, mice were treated with PBS, oxaliplatin, EVs with cetuximab-expressing membranes, or oxaliplatin-encapsulating EVs with cetuximab-expressing membranes at a dose of 5 mg/kg by intraperitoneal injection every three days. Tumor size was measured every two days until the end of the experiment on day 30. The mice were then sacrificed and the tumors were collected for observation. The tumor volume was calculated using the formula, tumor volume [mm 3 ] = (length [2]) × (width [2]) 2 × 0.52. The Kaohsiung Medical University Institutional Animal Care and Use Committee approved this study (approval no. 112056). All experimental procedures were conducted in accordance with regulations. Discussion In this study, we successfully established a stable system for secretion of EVs with cetuximab-expressing membranes and achieved passive encapsulation of oxaliplatin to form oxaliplatin-encapsulating EVs with cetuximab-expressing membranes. This approach maintained the particle size of the EVs. Furthermore, we confirmed that EVs with cetuximab-expressing membranes were specifically internalized by EGFR + cells, while EGFR - cells did not show this phenomenon. In the cytotoxicity experiments, we found that oxaliplatin-encapsulating EVs with cetuximab-expressing membranes exhibited significant cytotoxic effects on EGFR + cells. Moreover, in the xenograft cancer animal model, we confirmed the specific accumulation of oxaliplatin-encapsulating EVs with cetuximab-expressing membranes in EGFR + cells and significant enhancement of the therapeutic efficacy of oxaliplatin against EGFR + cancer cells. Targeting is crucial when using EVs for cancer therapy. For instance, Liang et al. employed genetic engineering techniques to introduce aene fusion between the CD63 transmembrane protein and Apo-A1 sequence in 293T host cells. EVs derived from these cells through electroporation exhibited enhanced suppression of miR-26a in HepG2 cells. However, further validation of these findings through animal experimentation is lacking [21]. Ohno et al. demonstrated that EVs can effectively deliver microRNA (miRNA) to breast cancer cells expressing EGFR. By modifying donor cells to express a fusion of the transmembrane domain of platelet-derived growth factor receptor and the GE11 peptide, targeted delivery to breast cancer cells was achieved [22]. Shi et al. applied a similar approach to breast cancer expressing human epidermal growth factor receptor 2 (HER2). They designed EVs that displayed anti-human CD3 and anti-human HER2 antibodies, resulting in SMART-Exos that could dual-target CD3 and HER2 receptors in T cells in breast cancer. These engineered SMART-Exos demonstrated highly efficient and specific anti-tumor activity, both in vitro and in vivo, by redirecting and activating cytotoxic T cells to attack HER2-expressing breast cancer cells, thereby showcasing the potential of targeting breast cancer immunotherapy using endogenous EVs [17, 18, 23, 24]. In Cheng et al.’s study, monoclonal antibodies specific to T cell CD3 and cancer cell-associated EGFR were expressed. These antibodies not only induced crosslinking between T cells and EGFR-expressing breast cancer cells but also showed efficacy in both in vitro and in vivo settings. They highlighted a novel application of EVs in cancer immunotherapy and suggested a universal approach for developing new cell-free therapies [18]. In our study, we used a mouse xenograft model to confirm the efficacy of oxaliplatin-encapsulating EVs with cetuximab-expressing membranes against EGFR+ colorectal cancer. In addition, we found that these vesicles exhibited certain inhibitory effects on EGFR+ colorectal cancer, underscoring the importance of targeted EV delivery (Fig. 7). This study also presents a widely applicable drug delivery platform for EVs, offering a safer option for delivering clinical antibody drugs and chemotherapy agents. Extracellular vesicles with antibody-expressing membranes can enhance efficacy and mitigate toxicity in normal cells, thereby minimizing the side effects of chemotherapy drugs. Zheng et al.’s study used EVs derived from T cells expressing chimeric antigen receptors (CAR) (CAR-Exos). Paclitaxel was encapsulated in these CAR-Exos (PTX@CAR-Exos) and administered to lung cancer mouse models through inhalation. The results showed that inhaled PTX@CAR-Exos accumulated within the tumor area and significantly reduced tumor size, extended survival time, and exhibited lower toxicity [25–28]. Rezakhani et al.'s study used various EVs loaded with doxorubicin and paclitaxel to treat different types of cancer. EVs carrying doxorubicin effectively inhibited the proliferation of breast cancer cells and induced apoptosis, while mitigating doxorubicin's toxicity and drug resistance. Similarly, paclitaxel-loaded EVs enhanced the cytotoxic effect of paclitaxel on prostate cancer cells and alleviated its side effects and drug resistance [29–31]. Schindler et al. successfully generated EVs carrying doxorubicin and demonstrated that the EVs could be rapidly taken up by cells. These EVs redistributed doxorubicin from the endoplasmic reticulum to the cytoplasm and nucleus, enhancing doxorubicin's efficacy against various cell lines. Notably, compared to alternative doxorubicin delivery methods, EVs did not accumulate in the heart, potentially mitigating cardiac side effects [32]. Uslu et al. explored the impact of human-platelet-released EVs carrying doxorubicin on breast cancer. The results indicated that, in a short time frame, the EVs carrying doxorubicin significantly reduced the survival rate of MDA-MB-231 cells compared to the direct application of doxorubicin [33]. Zhou et al.'s study of cisplatin-carrying EVs demonstrated rapid uptake of the EVs by cells and redistribution of cisplatin into the cytoplasm and nucleus. This approach enhanced the efficacy of cisplatin against multiple cell lines. Importantly, compared to alternative cisplatin delivery methods, EVs did not accumulate in the heart, potentially mitigating cardiac side effects [34]. These findings suggest that the employment of EVs with antibody-expressing membranes as drug carriers can enhance therapeutic efficacy and reduce toxicity to normal cells, thereby minimizing side effects. The utilization of EVs holds profound significance in the realm of cancer treatment, with their targeting precision and payload adaptability playing pivotal roles in the formulation of diverse therapeutic strategies. Cheng et al.’s groundbreaking work examining T cells (Jurkat) and EGFR-positive breast cancer (MDA-MB-468) unveiled the potential of EVs engineered with αCD3/αEGFR on their surface, aptly named SMART-Exos. These constructs effectively instigated T cells to engage with EGFR-positive triple-negative breast cancer cells, thereby activating T cell cytotoxicity and facilitating infiltration into the tumor microenvironment, ultimately enhancing their therapeutic efficacy against cancer [18]. An investigation of HER2-positive breast cancer cells (SKBR3) adopted designed ankyrin repeat proteins as markers for EVs, coupled with the loading of Tpd50 siRNA to augment their targeted killing efficacy against breast cancer cells [35]. Moreover, in the context of hepatocellular carcinoma (HepG2) treatment, Liang et al. strategically employed ApoA-1 as a targeting marker, harnessing its potential to deliver miRNA-26a safely and specifically into hepatocellular carcinoma, thereby amplifying the therapeutic impact [21]. For adenocarcinoma, exemplified by human alveolar basal epithelial cells (A549), the incorporation of iRGD peptide as an exosomal targeting ligand, alongside KRAS siRNA as the payload, endowed these EVs with the unique ability to precisely target and treat lung cancer [36]. In the therapeutic landscape of colorectal cancer (HCT-116), the integration of the HER2-binding affinity body zHER fused to the N-terminus of LAMP-2 facilitated EVs with heightened binding affinity and selectivity. This strategic design allowed for the specific delivery of 5-FU and anti-miRNA-21 drugs to tumors expressing HER2, underscoring its potential therapeutic impact [37]. Notably, our study explored the application of oxaliplatin-loaded EVs expressing EGFR antibodies and substantiated their potential in augmenting specific targeting effects on colorectal cancer. All these studies offer profound insights into the nuanced aspects of exosomal directionality and payload flexibility within the context of cancer treatment, thereby laying a robust foundation for the development of future cancer therapies. Declarations The ethic approval statement The Kaohsiung Medical University Institutional Animal Care and Use Committee approved this study (approval no. 112056). All experimental procedures were conducted in accordance with regulations. The funding statement This study was financially supported by clinical research grants from Kaohsiung Armed Force General Hospital, Kaohsiung, Taiwan (No. MAB-112-802KB112577), the Ministry of Science and Technology, Taiwan (No. 110-2320-B-037 -027 -MY3); the KMU-KMUH Co-Project of Key Research (No. KMU-DK(A)113017); and the Research Foundation (No. PT111001 and No. PT111002) of Kaohsiung Medical University, Taiwan. We thank the Drug Development and Value Creation Research Center, Kaohsiung Medical University, Taiwan for instrumentation and equipment support. Author contributions Chih-Hung Chuang, A Chien, and Yi-Jung Huang: Conceptualization, data curation, and formal analysis. Shang-Tao Chien, and Yi-Jung Huang: Funding acquisition, investigation, and project administration. Ming-Yii Huang, Yi-Ping Fang, Shi-Wei Chao, and Chia-Tse Li: Methodology. Wun-Ya Jhang and Yun-Han Hsu: Resources, software, and supervision. Shuo-Hung Wang: Validation and visualization. Shang-Tao Chien, Yi-Jung Huang, and Chih-Hung Chuang: Writing—original draft. Ming-Yii Huang and Chih-Hung Chuang: Writing—review and editing. Competing interests The authors declare that they have no competing interests. References Zeng, Y., et al., Biological Features of Extracellular Vesicles and Challenges. Front Cell Dev Biol, 2022. 10 : p. 816698. Zhang, L.Y., et al., Membrane Derived Vesicles as Biomimetic Carriers for Targeted Drug Delivery System. Curr Top Med Chem, 2020. 20 (27): p. 2472-2492. Fu, P., et al., Extracellular vesicles as delivery systems at nano-/micro-scale. Adv Drug Deliv Rev, 2021. 179 : p. 113910. Kooijmans, S.A.A., et al., Modulation of tissue tropism and biological activity of exosomes and other extracellular vesicles: New nanotools for cancer treatment. Pharmacol Res, 2016. 111 : p. 487-500. Somiya, M., Y. Yoshioka, and T. Ochiya, Biocompatibility of highly purified bovine milk-derived extracellular vesicles. J Extracell Vesicles, 2018. 7 (1): p. 1440132. Wu, P., et al., Extracellular vesicles: A bright star of nanomedicine. Biomaterials, 2021. 269 : p. 120467. Zhu, L.P., et al., Hypoxia-elicited mesenchymal stem cell-derived exosomes facilitates cardiac repair through miR-125b-mediated prevention of cell death in myocardial infarction. Theranostics, 2018. 8 (22): p. 6163-6177. Tian, J., et al., Engineered Exosome for Drug Delivery: Recent Development and Clinical Applications. Int J Nanomedicine, 2023. 18 : p. 7923-7940. Chen, Z., et al., Encapsulation and assessment of therapeutic cargo in engineered exosomes: a systematic review. J Nanobiotechnology, 2024. 22 (1): p. 18. Rahbarghazi, R., et al., Tumor-derived extracellular vesicles: reliable tools for Cancer diagnosis and clinical applications. Cell Commun Signal, 2019. 17 (1): p. 73. Chang, W.H., R.A. Cerione, and M.A. Antonyak, Extracellular Vesicles and Their Roles in Cancer Progression. Methods Mol Biol, 2021. 2174 : p. 143-170. Hou, Y., J. Wang, and J. Wang, Engineered biomaterial delivery strategies are used to reduce cardiotoxicity in osteosarcoma. Front Pharmacol, 2023. 14 : p. 1284406. Liu, X., C. Xiao, and K. Xiao, Engineered extracellular vesicles-like biomimetic nanoparticles as an emerging platform for targeted cancer therapy. J Nanobiotechnology, 2023. 21 (1): p. 287. Stridfeldt, F., et al., Analyses of single extracellular vesicles from non-small lung cancer cells to reveal effects of epidermal growth factor receptor inhibitor treatments. Talanta, 2023. 259 : p. 124553. Kooijmans, S.A.A., et al., PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time. J Control Release, 2016. 224 : p. 77-85. Li, Y., et al., A33 antibody-functionalized exosomes for targeted delivery of doxorubicin against colorectal cancer. Nanomedicine, 2018. 14 (7): p. 1973-1985. Shi, X., et al., Genetically Engineered Cell-Derived Nanoparticles for Targeted Breast Cancer Immunotherapy. Mol Ther, 2020. 28 (2): p. 536-547. Cheng, Q., et al., Reprogramming Exosomes as Nanoscale Controllers of Cellular Immunity. J Am Chem Soc, 2018. 140 (48): p. 16413-16417. Chao, M.P., I.L. Weissman, and R. Majeti, The CD47-SIRPalpha pathway in cancer immune evasion and potential therapeutic implications. Curr Opin Immunol, 2012. 24 (2): p. 225-32. Lin, W.W., et al., Enhancement Effect of a Variable Topology of a Membrane-Tethered Anti-Poly(ethylene glycol) Antibody on the Sensitivity for Quantifying PEG and PEGylated Molecules. Anal Chem, 2017. 89 (11): p. 6082-6090. Liang, G., et al., Engineered exosome-mediated delivery of functionally active miR-26a and its enhanced suppression effect in HepG2 cells. Int J Nanomedicine, 2018. 13 : p. 585-599. Ohno, S., et al., Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol Ther, 2013. 21 (1): p. 185-91. Cheng, Q., X. Shi, and Y. Zhang, Reprogramming Exosomes for Immunotherapy. Methods Mol Biol, 2020. 2097 : p. 197-209. Ferreira, D., J.N. Moreira, and L.R. Rodrigues, New advances in exosome-based targeted drug delivery systems. Crit Rev Oncol Hematol, 2022. 172 : p. 103628. Zheng, W., et al., Inhalable CAR-T cell-derived exosomes as paclitaxel carriers for treating lung cancer. J Transl Med, 2023. 21 (1): p. 383. Lee, H.H., et al., Therapeutic effiacy of T cells expressing chimeric antigen receptor derived from a mesothelin-specific scFv in orthotopic human pancreatic cancer animal models. Neoplasia, 2022. 24 (2): p. 98-108. Wang, Y., et al., Chemokine Receptor CCR2b Enhanced Anti-tumor Function of Chimeric Antigen Receptor T Cells Targeting Mesothelin in a Non-small-cell Lung Carcinoma Model. Front Immunol, 2021. 12 : p. 628906. Wang, P., et al., Exosomes from M1-Polarized Macrophages Enhance Paclitaxel Antitumor Activity by Activating Macrophages-Mediated Inflammation. Theranostics, 2019. 9 (6): p. 1714-1727. Rezakhani, L., et al., Exosomes: special nano-therapeutic carrier for cancers, overview on anticancer drugs. Med Oncol, 2022. 40 (1): p. 31. Lennaard, A.J., et al., Optimised Electroporation for Loading of Extracellular Vesicles with Doxorubicin. Pharmaceutics, 2021. 14 (1). Sutaria, D.S., et al., Achieving the Promise of Therapeutic Extracellular Vesicles: The Devil is in Details of Therapeutic Loading. Pharm Res, 2017. 34 (5): p. 1053-1066. Schindler, C., et al., Exosomal delivery of doxorubicin enables rapid cell entry and enhanced in vitro potency. PLoS One, 2019. 14 (3): p. e0214545. Uslu, D., et al., Effect of platelet exosomes loaded with doxorubicin as a targeted therapy on triple-negative breast cancer cells. Mol Divers, 2022. Zhou, G., et al., Exosome Mediated Cytosolic Cisplatin Delivery Through Clathrin-Independent Endocytosis and Enhanced Anti-cancer Effect via Avoiding Endosome Trapping in Cisplatin-Resistant Ovarian Cancer. Front Med (Lausanne), 2022. 9 : p. 810761. Limoni, S.K., et al., Engineered Exosomes for Targeted Transfer of siRNA to HER2 Positive Breast Cancer Cells. Appl Biochem Biotechnol, 2019. 187 (1): p. 352-364. Tian, Y., et al., A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials, 2014. 35 (7): p. 2383-90. Liang, G., et al., Engineered exosomes for targeted co-delivery of miR-21 inhibitor and chemotherapeutics to reverse drug resistance in colon cancer. J Nanobiotechnology, 2020. 18 (1): p. 10. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 17 Aug, 2024 Read the published version in Cancer Nanotechnology → Version 1 posted Editorial decision: Revision requested 19 Jun, 2024 Reviews received at journal 19 Jun, 2024 Reviews received at journal 11 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 09 Jun, 2024 Reviews received at journal 15 May, 2024 Reviewers agreed at journal 05 May, 2024 Reviewers invited by journal 02 May, 2024 Submission checks completed at journal 22 Apr, 2024 Editor assigned by journal 22 Apr, 2024 First submitted to journal 19 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4291698","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":294014230,"identity":"eed138c9-10ec-42b9-a26f-ce703e116a20","order_by":0,"name":"Shang-Tao Chien","email":"","orcid":"","institution":"Kaohsiung Armed Forces General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shang-Tao","middleName":"","lastName":"Chien","suffix":""},{"id":294014231,"identity":"55e277fd-2e35-48cf-82d5-43b2c86ad077","order_by":1,"name":"Yi-Jung Huang","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi-Jung","middleName":"","lastName":"Huang","suffix":""},{"id":294014232,"identity":"0cd85857-e6fc-44e1-8bef-f73255c8c210","order_by":2,"name":"Ming-Yii Huang","email":"","orcid":"","institution":"Kaohsiung Medical University Hospital, Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ming-Yii","middleName":"","lastName":"Huang","suffix":""},{"id":294014233,"identity":"cd65979b-e111-40c2-a049-9053ab47f306","order_by":3,"name":"Yi-Ping Fang","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi-Ping","middleName":"","lastName":"Fang","suffix":""},{"id":294014234,"identity":"06abf142-d01b-4ead-8968-463e2d2feaf5","order_by":4,"name":"Shi-Wei Chao","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shi-Wei","middleName":"","lastName":"Chao","suffix":""},{"id":294014235,"identity":"aa9616f7-5fb7-43da-9659-27e23b22df2f","order_by":5,"name":"Chia-Tse Li","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chia-Tse","middleName":"","lastName":"Li","suffix":""},{"id":294014236,"identity":"ca3b973e-fe6a-436b-9ed4-3d63e96d968b","order_by":6,"name":"Wun-Ya Jhang","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wun-Ya","middleName":"","lastName":"Jhang","suffix":""},{"id":294014237,"identity":"4b525d55-0a28-48ce-b746-43ba0297738c","order_by":7,"name":"Yun-Han Hsu","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yun-Han","middleName":"","lastName":"Hsu","suffix":""},{"id":294014239,"identity":"74ea9943-c3e1-4579-92aa-6087481763b0","order_by":8,"name":"Shuo-Hung Wang","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuo-Hung","middleName":"","lastName":"Wang","suffix":""},{"id":294014241,"identity":"4ca38a59-c9b4-4e21-ac95-9890b6a66d54","order_by":9,"name":"Chih-Hung Chuang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYFCCg80PPlRI8PCD2AkFxOhgPNxmOOOMjZxkA0iLATFamI83SPO2pBkbHADxiNHC33awwYC34XDi5vOrEz88MGCQ5xc7gF+LxJmDDQ8kdxxO3Hbj7WYJoMMMZ85OIGDNDaAthmdAWs5uAGlJMLhNQIv8/YcNEoltQIfNOLv5B1FaDA4cbJA42Ab0Pn/vNuJsMTxwsM2wARjIEjd4t1kkGEgQ9ovcgeOPH/8BRWX/2c03f1TYyPNLE9CCABJglRLEKgcB/gOkqB4Fo2AUjIKRBAByMFE6iXZX7AAAAABJRU5ErkJggg==","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":true,"prefix":"","firstName":"Chih-Hung","middleName":"","lastName":"Chuang","suffix":""}],"badges":[],"createdAt":"2024-04-19 07:58:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4291698/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4291698/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12645-024-00284-0","type":"published","date":"2024-08-17T15:58:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55330756,"identity":"00ffd529-f687-4749-ad27-c49b7954aa32","added_by":"auto","created_at":"2024-04-25 19:27:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":48140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of extracellular vesicles with cetuximab-expressing membranes inducing cytotoxicity against EGFR\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e colorectal cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOxaliplatin was passively loaded into a stable cell line expressing cetuximab in the membrane to produce oxaliplatin-encapsulating extracellular vesicles with cetuximab-expressing membranes (M-C-293EVs/OXA) to increase the toxicity of oxaliplatin to EGFR\u003csup\u003e+\u003c/sup\u003e colorectal cancer.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4291698/v1/5ee0b0fb371be65efce0838c.jpg"},{"id":55331221,"identity":"469c61da-5e76-4680-b299-5e26ace196d0","added_by":"auto","created_at":"2024-04-25 19:35:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of extracellular vesicles with cetuximab-expressing membranes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eto collect cytoplasmic fragments and extracellular vesicles from both normal HEK293 cells and HEK293 cells with cetuximab-expressing membranes, and detect human Fab, Hsp 70, CD81, and CD9 using specific antibodies. 293: HEK293 cell line; M-C-293: HEK293 cell line with cetuximab-expressing membrane; EVs: extracellular vesicles.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4291698/v1/f982114980f15f4cbf2fc3d0.jpg"},{"id":55330758,"identity":"0b370186-c7e0-4669-947c-2278ff040ca7","added_by":"auto","created_at":"2024-04-25 19:27:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":235909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of oxaliplatin-loaded extracellular vesicles with cetuximab-expressing membranes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtracellular vesicles were isolated from supernatants of HEK293 cells, as well as from an oxaliplatin-treated HEK293 cell line with cetuximab-expressing membrane. The shape and particle size of these vesicles were analyzed using (A) transmission electron microscopy (TEM) and (B) nanoparticle tracking. To prepare for TEM, the vesicles were placed on copper grids, stained with uranyl acetate, and examined. 293-EVs: Extracellular vesicles of HEK293 cells; M-293EVs: extracellular vesicles of HEK293 cell line with cetuximab-expressing membrane; M-C-293EVs/OXA: oxaliplatin-loaded extracellular vesicles of the HEK293 cell line with cetuximab-expressing membrane.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4291698/v1/569a6e0fe0920e3713f9a43c.jpg"},{"id":55330760,"identity":"454f23a9-7798-4dd7-bfe5-02ded9d626f4","added_by":"auto","created_at":"2024-04-25 19:27:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":79332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInternalization of rhodamine B packaged in membrane expressing EGFR Ab exosome into the EGFR\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e colorectal cell line.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRhodamine B was electroporated into extracellular vesicles (EVs) or membranes expressing anti-EGFR antibodies using the ECM-2001 Hybridoma System. These samples were then reacted for 30 minutes, 60 minutes, or 90 minutes and detected in (A) HCT116 (EGFR\u003csup\u003e+\u003c/sup\u003e) or (B) SW620 (EGFR\u003csup\u003e-\u003c/sup\u003e) cells using fluorescent microscopy. The excitation/emission spectra of rhodamine B were 535/595–600 nm. The excitation/emission spectra of DAPI were 358/461 nm. The excitation/emission spectra of CMFDA were 492/517nm. 293-EVs: extracellular vesicles from HEK293 cell line with cetuximab-expressing membrane; M-C293EVs/Rho: rhodamine B-loaded extracellular vesicles from HEK293 cell line with cetuximab-expressing membrane.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4291698/v1/40bb750b6b3f01bb6e8a88e0.jpg"},{"id":55330757,"identity":"5a0f9426-9ad7-415d-950b-564e4de30866","added_by":"auto","created_at":"2024-04-25 19:27:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":40571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxaliplatin-loaded extracellular vesicles with cetuximab-expressing membranes increase the cytotoxicity of oxaliplatin against colorectal cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerially diluted extracellular vesicles, oxaliplatin-loaded extracellular vesicles, extracellular vesicles with cetuximab-expressing membranes, or oxaliplatin-loaded extracellular vesicles with cetuximab-expressing membranes were used to treat HCT116 and SW620 cell lines. After 90 minutes of reaction, the culture medium was removed and new culture medium was added for 48 hours for further reaction. Cell activity was analyzed using ATPlite (A, B). EVs: extracellular vesicles from the HEK293 cell line; EVs/OXA: oxaliplatin-loaded extracellular vesicles from the HEK293 cell line M-C-293EVs: extracellular vesicles produced by HEK293 cell line with cetuximab-expressing membrane; M-C-293EVs/OXA: oxaliplatin-loaded extracellular vesicles produced by HEK293 cell line with cetuximab-expressing membrane.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4291698/v1/1263399953277d8e5bc7280c.jpg"},{"id":55330762,"identity":"edc553d5-b3af-4eb6-b8eb-cd9d9624dbe6","added_by":"auto","created_at":"2024-04-25 19:27:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":208939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe specificity of accumulation of rhodamine B-loaded extracellular vesicles with cetuximab-expressing membranes in cancer cells in the xenograft model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdministration of (A) 2 mg/kg of rhodamine B-loaded extracellular vesicles and (B) 2 mg/kg of rhodamine B-loaded extracellular vesicles with cetuximab-expressing membranes separately via injection into the tail vein of mice bearing SW620 (left leg) and HCT116 (right leg) tumors. Monitoring of accumulation of rhodamine B (Em: 555 nm; Ex: 580 nm) at different time points using fluorescent IVIS images. 293EVs/Rho: rhodamine-loaded extracellular vesicles from HEK293 cell line; M-C-293EVs/Rho: rhodamine-loaded extracellular vesicles from HEK293 cell line with cetuximab-expressing membrane.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4291698/v1/dfaacea6930ffc8e34f8426e.jpg"},{"id":55330759,"identity":"05e65174-2101-423a-91bd-2f41431813e9","added_by":"auto","created_at":"2024-04-25 19:27:47","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":46396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxaliplatin-encapsulating extracellular vesicles with cetuximab-expressing membranes enhance the therapeutic efficacy of oxaliplatin against EGFR\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e colorectal cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePBS, oxaliplatin (OXA), extracellular vesicles from the HEK293 cell line with cetuximab-expressing membrane (M-C-293EVs), or oxaliplatin-encapsulating extracellular vesicles from the HEK293 cell line with cetuximab-expressing membrane (M-C-293EVs/OXA) were injected into the tail vein, once per three days, three times in total. The tumor volume was measured in the (A) HCT116 and (B) SW620 xenograft models.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4291698/v1/2e90f0e41176d796dc216860.jpg"},{"id":63071642,"identity":"bb051be9-e98d-4220-a75a-6217b1957a54","added_by":"auto","created_at":"2024-08-22 20:09:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1679971,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4291698/v1/c871e5a7-75ed-4d0a-8d1d-f1ad44daa4ec.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Maximizing Oxaliplatin's Impact on EGFR+ Colorectal Cancer Through Targeted Extracellular Vesicles","fulltext":[{"header":"Highlights","content":"\u003col\u003e\n \u003cli\u003eEVs with cetuximab-expressing membranes were used to encapsulate oxaliplatin and target EGFR\u003csup\u003e+\u003c/sup\u003e cells.\u003c/li\u003e\n \u003cli\u003eOxaliplatin-loaded EVs\u0026nbsp;with cetuximab-expressing membranes exhibited significant cytotoxic effects on EGFR\u003csup\u003e+\u003c/sup\u003e cells.\u003c/li\u003e\n \u003cli\u003eIn a xenograft cancer animal model, oxaliplatin-loaded EVs with cetuximab-expressing membranes accumulated specifically in EGFR\u003csup\u003e+\u003c/sup\u003e cells and significantly enhanced oxaliplatin\u0026rsquo;s therapeutic efficacy against EGFR+ cancer cells.\u003c/li\u003e\n \u003cli\u003eEVs with targeted membrane expression of drugs are a promising strategy for delivering therapeutic agents to specific cell populations.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eExtracellular vesicles (EVs) are secreted by the cells themselves, which makes them biocompatible. Compared to synthetic drug carriers, EVs undergo more natural excretion and metabolism processes in the body. In addition, EVs can reduce interactions with the immune system, thereby mitigating potential immune reactions. These characteristics make them excellent carriers for the delivery of therapeutic drugs\u0026nbsp;[1\u0026ndash;4]. For example, Somiya et al. demonstrated excellent biocompatibility of EVs in vivo, as they were cleared without adverse reactions.\u0026nbsp;The toxicity, immunology, and immunogenicity of EVs have also been evaluated, with results showing acceptable biocompatibility in vivo\u0026nbsp;[5]. Wu et al. highlighted that EVs, being products of endogenous secretion, exhibit good biocompatibility within the body and can reduce interactions with the immune system. These attributes confer potential advantages to EVs as drug delivery systems and mitigate the risk of immune reactions\u0026nbsp;[6].\u003c/p\u003e\n\u003cp\u003eTargeting the specificity of EVs is crucial for their application in disease treatment. For instance, Zhu et al. demonstrated that conjugated Hypo-Exo with an ischemic myocardium-targeted peptide facilitates ischemic cardiac repair by ameliorating cardiomyocyte apoptosis. This indicates that particular peptides can enable EVs to acquire targeted specificity [7]. In breast cancer treatment, the targeting of EVs using anti-human CD3 and anti-human HER2 antibodies has been employed to generate SMART-Exos that dual-target T cells to CD3 and HER2 receptors associated with breast cancer. This approach enhances the efficacy of targeting of HER2-positive breast cancer cells by T cells [8\u0026ndash;13]. In lung cancer treatment, ligands that bind to the epidermal growth factor receptor (EGFR) can be modified to attach to the surface of EVs to increase their binding and uptake by EGFR-positive tumor cells. In previous studies, EVs have been co-incubated with epidermal growth factor or EGFR antibodies to allow the ligands to bind to the EV surface and the targeted effects of the modified EVs on tumor cells have been evaluated using cell-based assays and animal models [14\u0026ndash;19]. Although targeted EVs have been applied in breast cancer and lung cancer, their application in colorectal cancer remains unclear.\u003c/p\u003e\n\u003cp\u003eIn this study, we passively loaded oxaliplatin into a stable cell line expressing cetuximab, an EGFR inhibitor, in the membrane and collected its EVs to confirm their ability to load oxaliplatin and target EGFR to increase the cytotoxicity of oxaliplatin in EGFR\u003csup\u003e+\u003c/sup\u003e colorectal cancer cells (Fig. 1). We constructed EVs able to express cetuximab on the membrane and then confirmed cetuximab expression on the EV membrane. We also confirmed whether the EVs changed their particle size after encapsulating oxaliplatin. We investigated whether EVs with cetuximab-expressing membranes could be internalized by EGFR\u003csup\u003e+\u003c/sup\u003e colorectal cancer cells to increase cytotoxicity. Finally, we established a xenograft colorectal animal model to confirm the tumor-specific accumulation of oxaliplatin-encapsulating EVs with cetuximab-expressing membranes and their therapeutic effect on EGFR\u003csup\u003e+\u0026nbsp;\u003c/sup\u003ecolorectal cancer.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExtracellular vesicles\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;with cetuximab-expressing membranes were successfully constructed\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;To enable cetuximab expression on the membranes of EVs, we constructed stable HEK cell lines with their membranes expressing the cetuximab antibody. We cultured\u0026nbsp;HEK293\u0026nbsp;cells and HEK293 cells secreting EVs with cetuximab-expressing membranes. We then collected the cell supernatant and subjected it to differential centrifugation to isolate HEK293 EVs and HEK293 EVs with cetuximab-expressing membranes. Western blot analysis was performed to confirm the expression of cetuximab in HEK293 cells, in HEK293 cells with cetuximab-expressing membranes, in HEK293 EVs, and in HEK293 EVs with cetuximab-expressing membranes, as well as to assess the accuracy of our sample\u0026nbsp;collection. In addition, human Fab analysis was performed to confirm successful cetuximab expression. We also analyzed specific markers of EVs, such as Hsp70, CD81, and CD9. The results indicated the presence of their respective specific molecules in EVs compared to the cell groups, demonstrating successful collection of EVs from both HEK293 cells and HEK293 cells with cetuximab-expressing membranes. Further comparison of the expression levels of cetuximab in the EVs of HEK293 cells and the EVs of HEK293 cells with cetuximab-expressing membranes showed that both the cells and their EVs expressed the human antibody, indicating successful establishment of the HEK293 cell line with cetuximab-expressing membranes and EVs with cetuximab-expressing membranes (Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtracellular vesicles with cetuximab-expressing membranes were successfully loaded with oxaliplatin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;To confirm the successful encapsulation of oxaliplatin by EVs with cetuximab-expressing membranes, we introduced oxaliplatin into one group of cells secreting EVs with membrane-expressed cetuximab, while another group of similar cells was not treated with oxaliplatin. UV irradiation was then applied for 30 min, followed by the collection of EVs. After subjecting the collected EVs to high-speed centrifugation, we analyzed them using transmission electron microscopy (Fig. 3A) and nanoparticle tracking (Fig. 3B). The results of transmission electron microscopy showed that the particles of oxaliplatin-encapsulating EVs with cetuximab-expressing membranes were slightly larger than similar vesicles without oxaliplatin encapsulation (152.8 \u0026plusmn; 1.4 nm vs. 142.4 \u0026plusmn; 1.2 nm). However, the nanoparticle tracking analysis did not find a significant difference in particle size. These results confirm the successful establishment of oxaliplatin-encapsulating EVs with cetuximab-expressing membranes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtracellular vesicles with cetuximab-expressing membranes can undergo EGFR\u003csup\u003e+\u003c/sup\u003e colorectal cancer-specific internalization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;To confirm whether EVs with cetuximab-expressing membranes can undergo EGFR+ colorectal cancer-specific internalization, we coated rhodamine B on EVs with normal membranes and those with membranes expressing cetuximab. These vesicles were separately added to HCT116 (EGFR\u003csup\u003e+\u003c/sup\u003e) and SW620 (EGFR\u003csup\u003e-\u003c/sup\u003e) cells and incubated for a specific period. Afterward, the vesicles that did not bind or were not internalized into the cells were washed away with PBS. The results showed significant internalization in the HCT116 (EGFR\u003csup\u003e+\u003c/sup\u003e) group compared to the SW620 (EGFR\u003csup\u003e-\u003c/sup\u003e) group (Fig. 4A vs. Fig. 4B). In addition, in the comparison of the cell-derived vesicles with membranes able to express cetuximab and those without in the HCT116 group, the EVs with cetuximab-expressing membranes exhibited more pronounced internalization (Fig. 4A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxaliplatin-encapsulating extracellular vesicles\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;with cetuximab-expressing membranes increase the cytotoxicity of oxaliplatin against colorectal cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;To confirm whether oxaliplatin-encapsulating EVs with cetuximab-expressing membranes can enhance the cytotoxicity of oxaliplatin against EGFR\u003csup\u003e+\u003c/sup\u003e colorectal cancer cells, we separately cultured HCT116 and SW620 cells and treated them with EVs encapsulating oxaliplatin, or EVs with cetuximab-expressing membranes, or oxaliplatin-encapsulating EVs with cetuximab-expressing membranes. After 1 hour, the cells were washed with PBS and incubated in fresh culture medium for 48 hours. Cell viability was then analyzed. The results showed that compared to oxaliplatin-encapsulating EVs with normal membranes, oxaliplatin-encapsulating EVs with cetuximab-expressing membranes exhibited higher cytotoxicity in EGFR\u003csup\u003e+\u003c/sup\u003e cells (Fig. 5). However, this phenomenon was not observed in EGFR\u003csup\u003e-\u003c/sup\u003e cells. Based on these findings, we suggest that oxaliplatin-encapsulating EVs with cetuximab-expressing membranes can enhance the killing effect of oxaliplatin specifically on EGFR\u003csup\u003e+\u003c/sup\u003e cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe specificity of accumulation of rhodamine B-loaded extracellular vesicles with\u0026nbsp;cetuximab-expressing membranes\u0026nbsp;in cancer cells in a xenograft model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;We performed an additional experiment to confirm the specific accumulation of EVs with membrane-expressed cetuximab in tumor cells. We injected SW620 cells into the left leg and HCT116 cells into the right leg of mice, allowing them to grow into a tumor sized 50 mm\u003csup\u003e3\u003c/sup\u003e. Then, we administered rhodamine B-loaded EVs with normal membranes and rhodamine B-loaded EVs with cetuximab-expressing membranes into the tail vein. We determined the absorbance of a 580-nm wavelength at 0 min, 30 min, 120 min, 180 min, 240 min, and 360 min. The results indicated that compared to the group treated with EVs with normal membranes, the group treated with EVs with cetuximab-expressing membranes showed significant accumulation of rhodamine B in the tumor. Furthermore, compared to the fluorescence accumulation in SW620 cells (Fig. 6A), the fluorescence accumulation in HCT116 cells was higher after 120 min (Fig. 6B). This phenomenon suggests that membrane-expressed cetuximab can selectively deliver compounds to target tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe therapeutic efficacy of oxaliplatin against EGFR\u003csup\u003e+\u003c/sup\u003e colorectal cancer is enhanced when it is encapsulated in extracellular vesicles with cetuximab-expressing membranes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;To assess the treatment efficacy against EGFR\u003csup\u003e+\u003c/sup\u003e colorectal cancer of oxaliplatin encapsulated in EVs with cetuximab-expressing membranes, we subcutaneously injected SW620 and HCT116 cells into the left legs of mice. Once the tumors reached a volume of 50 mm\u003csup\u003e3\u003c/sup\u003e, we administered PBS or 5 mg/kg/3 days (cumulative 3 doses) of oxaliplatin, EVs with cetuximab-expressing membranes, or oxaliplatin-encapsulating EVs with cetuximaexpressing membranes. Tumor volume was measured every three days until day 30. In the HCT116 tumor model, the results showed that the tumor volume was smaller in the group treated with EVs with cetuximab-expressing membranes compared to the PBS group, and the tumor volume was even smaller in the group treated with oxaliplatin-encapsulating EVs with cetuximab-expressing membranes compared to the oxaliplatin group (Fig. 7A). However, no significant differences were observed in the SW620 tumor model (Fig. 7B). These results indicate that oxaliplatin-encapsulating EVs with cetuximab-expressing membranes had the highest efficacy against HCT116 tumor cells (Fig. 7A).\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell lines and cell culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;HEK293, HCT116 (EGFR\u003csup\u003e+\u003c/sup\u003e), and SW620 (EGFR\u003csup\u003e-\u003c/sup\u003e) cell lines (Bioresource Collection and Research Center, Hsinchu 300193, Taiwan) were cultured in Dulbecco\u0026apos;s Modified Eagle Medium (Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% bovine calf serum (Cytiva, Washington, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific Inc., Waltham, MA, USA). The cells were maintained in an incubator with 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eat 37 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of extracellular vesicles with membrane-expressed cetuximab and oxaliplatin encapsulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The light and heavy chains of cetuximab (NCBI, US) fused to the immunoglobulin C2-type extracellular transmembrane\u0026ndash;cytosolic domains of the mouse B7-1 antigen (eB7)\u0026nbsp;[20]\u0026nbsp;were cloned into the pLKO_AS2 vector (National Core Facility for Biopharmaceuticals, Taipei, Taiwan) using NheI, AscI, BglII, and BstxI restriction enzymes. The plasmid was then transformed into TOP 10 cells (Thermo Fisher Scientific Inc.) for amplification and extraction. The lentiviral vector was produced by co-transfecting pMD.G, pCMV\u0026Delta;R8.91 (National Core Facility for Biopharmaceuticals), and pLKO_AS2 cetuximab into HEK293 cells. Stable HEK293 cell lines that can secrete EVs with cetuximab-expressing membranes were selected using 1\u0026nbsp;mg/ml of neomycin sulfate (Thermo Fisher Scientific Inc.) after infection with virus. The HEK293 cells were cultured in serum-free medium and treated with 5mg/ml of oxaliplatin (Merck, Darmstadt, Germany) and irradiated with UVB once at a dose of 30 mJ/cm\u003csup\u003e2\u003c/sup\u003e for 24 hours The EVs with cetuximab-expressing membranes were collected from the cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection of extracellular vesicles with cetuximab-expressing membranes and Western blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Collecting EVs from drug-treated or untreated cell lines involved a series of centrifugation steps. The conditioned medium from the cells was first centrifuged at 300 \u0026times; g for 10 min at 4 \u0026deg;C to remove cellular debris. The supernatant was then subjected to further centrifugation at 2000 \u0026times; g for 10 min at 4 \u0026deg;C to remove larger microvesicles. Next, the supernatant was centrifuged at 10,000 \u0026times; g for 30 min at 4 \u0026deg;C to pellet smaller EVs. Finally, the pellet was resuspended in physiological saline and subjected to ultracentrifugation at 100,000 \u0026times; g for 1.5 hours at 4 \u0026deg;C to collect the extracellular vesicle pellet. For Western blot analysis, the collected EVs with cetuximab-expressing membranes, at 10 ug/well, were separated by SDS-PAGE and transferred onto a nitrocellulose NC membrane (Schleicher and Schuell, Einbeck, Germany). The NC membranes were probed with Mouse Anti-Human IgG Fab Antibody [HRP] (GenScript Biotech, Piscataway, NJ, USA), monoclonal HSP70 antibody (3A3, MA3-006; Thermo Fisher Scientific Inc.), anti-CD81 antibody (ab155760; abcam, Cambridge, UK), CD9 (D8O1A) Rabbit mAb #13174 (abcam), peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG, Fc Fragment Specific (115-035-008, 1:1000; Jackson ImmunoResearch, West Grove, PA, USA), or Rabbit Anti-Goat IgG Antibody, HRP conjugate, (rabbit IgG, AP106P, 1:1000; Sigma-Aldrich) and visualized with the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500; Merck, Rahway, NJ, USA), according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy and nanoparticle tracking analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Two micrograms of EVs\u0026nbsp;were pipetted (5 ml) onto formvar-coated copper grids (FF200-Cu; Electron Microscopy Sciences, Hatfield, PA, USA) and allowed to settle for 20 min at room temperature. Excess phosphate-buffered saline (PBS) was removed by wicking with filter paper before fixation using a 2% paraformaldehyde, 2% glutaraldehyde, and 0.05 M phosphate solution for 2 min. Grids were washed three times with distilled water prior to application of 1% phosphotungstic acid counterstain for 1 min. Excess liquid was removed by wicking with filter paper, and the grids were allowed to dry overnight at room temperature. Grids were analyzed using a transmission electron microscope (Technai G2 20; FEI, Hillsboro, OR, USA).\u0026nbsp;EVs were visualized and quantified using a nanoparticle tracking analyzer (Nanosight NS300; Nanosight Ltd., Amesbury, UK) with 70 mW laser with a wavelength of 405 nm. Urine samples were diluted in PBS at a ratio of 1:100, while serum samples were diluted at 1:1000. Duplicate measurements were recorded for each sample.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRhodamine B packaging by extracellular vesicles with cetuximab-expressing membranes and fluorescent staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;After mixing 5 mg/ml of rhodamine B (TCI,\u0026nbsp;Tokyo,\u0026nbsp;Japan) with 2 mg/ml of EVs with cetuximab-expressing membranes, the reaction was allowed to proceed for 1 hour. The mixture was then washed with PBS to separate the free rhodamine B and EVs by centrifugation at 100,000\u0026nbsp;\u0026times;\u0026nbsp;g for 10 min at 4 \u0026deg;C in an ultracentrifuge (Optima\u0026trade; XPN; Beckman Coulter,\u0026nbsp;Brea, California, USA). The pellet was collected and resuspended in PBS for further use. Then, 2\u0026nbsp;\u0026times;\u0026nbsp;10\u003csup\u003e3\u003c/sup\u003e SW620 and HCT116 cells were separately seeded on glass slides and 5 \u0026mu;M of rhodamine B-loaded EVs with cetuximab-expressing membranes was added to each cell culture and incubated for 90 min. The supernatant was removed and cells were washed with PBS. The cells were then fixed with 10% formalin (KINGFEX CO., LTD., Taipei, Taiwan) and stained with CellTracker\u0026trade; Green CMFDA Dye (Thermo Fisher Scientific Inc.) and DAPI (Merck, Darmstadt, Germany) and mounted with mounting media (Thermo Fisher Scientific Inc.). Fluorescent images were acquired with a confocal fluorescence microscope (FV1000; Olympus,\u0026nbsp;Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cells were plated in 96-well plates (2000 cells/well) overnight and serially diluted EVs, oxaliplatin-encapsulating EVs, EVs with cetuximab-expressing membranes, or oxaliplatin-encapsulating EVs with cetuximab-expressing membranes were added.Cell viability was detected using the ATPlite kit (510-17281; PerkinElmer, Chennai, India) and the luminesce value was measured using a multimode plate reader (VICTORTM X2; PerkinElmer).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of an ectopic cancer mouse model and analysis of the accumulation of rhodamine B-loaded extracellular vesicles with cetuximab-expressing membranes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Eight-week-old male nude mice (BALB/cAnN.Cg-Foxn1nu/CrlNarl) obtained from the National Laboratory Animal Center, Taiwan, were used in this study. The HCT116 and SW620 cell suspensions (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e in PBS) were subcutaneously inoculated into the right hind leg of the mice. Tumors were allowed to grow until they reached approximately 50 mm\u003csup\u003e3\u0026nbsp;\u003c/sup\u003ein size. Rhodamine B-loaded EVs were injected into the tail vein at a dose of 5 mg/kg, and the accumulation of tumor fluorescence was measured at different time points using IVIS (PerkinElmer, Inc., Waltham, MA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxaliplatin-encapsulating extracellular vesicles with cetuximab-expressing membranes enhance the therapeutic effect of oxaliplatin on EGFR+ cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;When the tumors reached approximately 50 mm\u003csup\u003e3\u003c/sup\u003e in size, mice were treated with PBS, oxaliplatin, EVs with cetuximab-expressing membranes, or oxaliplatin-encapsulating EVs with cetuximab-expressing membranes at a dose of 5 mg/kg by intraperitoneal injection every three days. Tumor size was measured every two days until the end of the experiment on day 30. The mice were then sacrificed and the tumors were collected for observation. The tumor volume was calculated using the formula, tumor volume [mm\u003csup\u003e3\u003c/sup\u003e] = (length [2]) \u0026times; (width [2])\u003csup\u003e2\u003c/sup\u003e \u0026times; 0.52. The Kaohsiung Medical University Institutional Animal Care and Use Committee approved this study (approval no. 112056). All experimental procedures were conducted in accordance with regulations.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we successfully established a stable system for secretion of EVs with cetuximab-expressing membranes and achieved passive encapsulation of oxaliplatin to form oxaliplatin-encapsulating EVs with cetuximab-expressing membranes. This approach maintained the particle size of the EVs. Furthermore, we confirmed that EVs with cetuximab-expressing membranes were specifically internalized by EGFR\u003csup\u003e+\u003c/sup\u003e cells, while EGFR\u003csup\u003e-\u003c/sup\u003e cells did not show this phenomenon. In the cytotoxicity experiments, we found that oxaliplatin-encapsulating EVs with cetuximab-expressing membranes exhibited significant cytotoxic effects on EGFR\u003csup\u003e+\u003c/sup\u003e cells. Moreover, in the xenograft cancer animal model, we confirmed the specific accumulation of oxaliplatin-encapsulating EVs with cetuximab-expressing membranes in EGFR\u003csup\u003e+\u003c/sup\u003e cells and significant enhancement of the therapeutic efficacy of oxaliplatin against EGFR\u003csup\u003e+\u003c/sup\u003e cancer cells.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Targeting is crucial when using EVs for cancer therapy. For instance, Liang et al. employed genetic engineering techniques to introduce aene fusion between the CD63 transmembrane protein and Apo-A1 sequence in 293T host cells. EVs derived from these cells through electroporation exhibited enhanced suppression of miR-26a in HepG2 cells. However, further validation of these findings through animal experimentation is lacking\u0026nbsp;[21]. Ohno et al. demonstrated that EVs can effectively deliver microRNA (miRNA) to breast cancer cells expressing EGFR. By modifying donor cells to express a fusion of the transmembrane domain of platelet-derived growth factor receptor and the GE11 peptide, targeted delivery to breast cancer cells was achieved\u0026nbsp;[22]. Shi et al. applied a similar approach to breast cancer expressing human epidermal growth factor receptor 2 (HER2). They designed EVs that displayed anti-human CD3 and anti-human HER2 antibodies, resulting in SMART-Exos that could dual-target CD3 and HER2 receptors in T cells in breast cancer. These engineered SMART-Exos demonstrated highly efficient and specific anti-tumor activity, both in vitro and in vivo, by redirecting and activating cytotoxic T cells to attack HER2-expressing breast cancer cells, thereby showcasing the potential of targeting breast cancer immunotherapy using endogenous EVs\u0026nbsp;[17, 18, 23, 24]. In Cheng et al.\u0026rsquo;s study, monoclonal antibodies specific to T cell CD3 and cancer cell-associated EGFR were expressed. These antibodies not only induced crosslinking between T cells and EGFR-expressing breast cancer cells but also showed efficacy in both in vitro and in vivo settings. They highlighted a novel application of EVs in cancer immunotherapy and suggested a universal approach for developing new cell-free therapies\u0026nbsp;[18]. In our study, we used a mouse xenograft model to confirm the efficacy of oxaliplatin-encapsulating EVs with cetuximab-expressing membranes against EGFR+ colorectal cancer. In addition, we found that these vesicles exhibited certain inhibitory effects on EGFR+ colorectal cancer, underscoring the importance of targeted EV delivery (Fig. 7). This study also presents a widely applicable drug delivery platform for EVs, offering a safer option for delivering clinical antibody drugs and chemotherapy agents.\u003c/p\u003e\n\u003cp\u003eExtracellular vesicles with antibody-expressing membranes can enhance efficacy and mitigate toxicity in normal cells, thereby minimizing the side effects of chemotherapy drugs. Zheng et al.\u0026rsquo;s study used EVs derived from T cells expressing chimeric antigen receptors (CAR) (CAR-Exos). Paclitaxel was encapsulated in these CAR-Exos (PTX@CAR-Exos) and administered to lung cancer mouse models through inhalation. The results showed that inhaled PTX@CAR-Exos accumulated within the tumor area and significantly reduced tumor size, extended survival time, and exhibited lower toxicity\u0026nbsp;[25\u0026ndash;28]. Rezakhani et al.\u0026apos;s study used various EVs loaded with doxorubicin and paclitaxel to treat different types of cancer. EVs carrying doxorubicin effectively inhibited the proliferation of breast cancer cells and induced apoptosis, while mitigating doxorubicin\u0026apos;s toxicity and drug resistance. Similarly, paclitaxel-loaded EVs enhanced the cytotoxic effect of paclitaxel on prostate cancer cells and alleviated its side effects and drug resistance\u0026nbsp;[29\u0026ndash;31]. Schindler et al. successfully generated EVs carrying doxorubicin and demonstrated that the EVs could be rapidly taken up by cells. These EVs redistributed doxorubicin from the endoplasmic reticulum to the cytoplasm and nucleus, enhancing doxorubicin\u0026apos;s efficacy against various cell lines. Notably, compared to alternative doxorubicin delivery methods, EVs did not accumulate in the heart, potentially mitigating cardiac side effects\u0026nbsp;[32]. Uslu et al. explored the impact of human-platelet-released EVs carrying doxorubicin on breast cancer. The results indicated that, in a short time frame, the EVs carrying doxorubicin significantly reduced the survival rate of MDA-MB-231 cells compared to the direct application of doxorubicin\u0026nbsp;[33]. Zhou et al.\u0026apos;s study of cisplatin-carrying EVs demonstrated rapid uptake of the EVs by cells and redistribution of cisplatin into the cytoplasm and nucleus. This approach enhanced the efficacy of cisplatin against multiple cell lines. Importantly, compared to alternative cisplatin delivery methods, EVs did not accumulate in the heart, potentially mitigating cardiac side effects\u0026nbsp;[34]. These findings suggest that the employment of EVs with antibody-expressing membranes as drug carriers can enhance therapeutic efficacy and reduce toxicity to normal cells, thereby minimizing side effects.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; The utilization of EVs holds profound significance in the realm of cancer treatment, with their targeting precision and payload adaptability playing pivotal roles in the formulation of diverse therapeutic strategies. Cheng et al.\u0026rsquo;s groundbreaking work examining T cells (Jurkat) and EGFR-positive breast cancer (MDA-MB-468) unveiled the potential of EVs engineered with \u0026alpha;CD3/\u0026alpha;EGFR on their surface, aptly named SMART-Exos. These constructs effectively instigated T cells to engage with EGFR-positive triple-negative breast cancer cells, thereby activating T cell cytotoxicity and facilitating infiltration into the tumor microenvironment, ultimately enhancing their therapeutic efficacy against cancer [18]. An investigation of HER2-positive breast cancer cells (SKBR3) adopted designed ankyrin repeat proteins as markers for EVs, coupled with the loading of Tpd50 siRNA to augment their targeted killing efficacy against breast cancer cells [35]. Moreover, in the context of hepatocellular carcinoma (HepG2) treatment, Liang et al. strategically employed ApoA-1 as a targeting marker, harnessing its potential to deliver miRNA-26a safely and specifically into hepatocellular carcinoma, thereby amplifying the therapeutic impact [21]. For adenocarcinoma, exemplified by human alveolar basal epithelial cells (A549), the incorporation of iRGD peptide as an exosomal targeting ligand, alongside KRAS siRNA as the payload, endowed these EVs with the unique ability to precisely target and treat lung cancer [36]. In the therapeutic landscape of colorectal cancer (HCT-116), the integration of the HER2-binding affinity body zHER fused to the N-terminus of LAMP-2 facilitated EVs with heightened binding affinity and selectivity. This strategic design allowed for the specific delivery of 5-FU and anti-miRNA-21 drugs to tumors expressing HER2, underscoring its potential therapeutic impact [37]. Notably, our study explored the application of oxaliplatin-loaded EVs expressing EGFR antibodies and substantiated their potential in augmenting specific targeting effects on colorectal cancer. All these studies offer profound insights into the nuanced aspects of exosomal directionality and payload flexibility within the context of cancer treatment, thereby laying a robust foundation for the development of future cancer therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eThe ethic approval statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Kaohsiung Medical University Institutional Animal Care and Use Committee approved this study (approval no. 112056). All experimental procedures were conducted in accordance with regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe funding statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by clinical research grants from Kaohsiung Armed Force General Hospital, Kaohsiung, Taiwan (No. MAB-112-802KB112577), the Ministry of Science and Technology, Taiwan (No. 110-2320-B-037 -027 -MY3); the KMU-KMUH Co-Project of Key Research (No. KMU-DK(A)113017); and the Research Foundation (No. PT111001 and\u0026nbsp;No. PT111002) of Kaohsiung Medical University, Taiwan. We thank the Drug Development and Value Creation Research Center, Kaohsiung Medical University, Taiwan for instrumentation and equipment support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChih-Hung Chuang, A Chien, and Yi-Jung Huang: Conceptualization, data curation, and formal analysis. Shang-Tao Chien, and Yi-Jung Huang: Funding acquisition, investigation, and project administration. Ming-Yii Huang, Yi-Ping Fang, Shi-Wei Chao, and Chia-Tse Li: Methodology. Wun-Ya Jhang and Yun-Han Hsu: Resources, software, and supervision. Shuo-Hung Wang: Validation and visualization. Shang-Tao Chien, Yi-Jung Huang, and Chih-Hung Chuang: Writing\u0026mdash;original draft. Ming-Yii Huang and Chih-Hung Chuang: Writing\u0026mdash;review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZeng, Y., et al., \u003cem\u003eBiological Features of Extracellular Vesicles and Challenges.\u003c/em\u003e Front Cell Dev Biol, 2022. \u003cstrong\u003e10\u003c/strong\u003e: p. 816698.\u003c/li\u003e\n\u003cli\u003eZhang, L.Y., et al., \u003cem\u003eMembrane Derived Vesicles as Biomimetic Carriers for Targeted Drug Delivery System.\u003c/em\u003e Curr Top Med Chem, 2020. \u003cstrong\u003e20\u003c/strong\u003e(27): p. 2472-2492.\u003c/li\u003e\n\u003cli\u003eFu, P., et al., \u003cem\u003eExtracellular vesicles as delivery systems at nano-/micro-scale.\u003c/em\u003e Adv Drug Deliv Rev, 2021. \u003cstrong\u003e179\u003c/strong\u003e: p. 113910.\u003c/li\u003e\n\u003cli\u003eKooijmans, S.A.A., et al., \u003cem\u003eModulation of tissue tropism and biological activity of exosomes and other extracellular vesicles: New nanotools for cancer treatment.\u003c/em\u003e Pharmacol Res, 2016. \u003cstrong\u003e111\u003c/strong\u003e: p. 487-500.\u003c/li\u003e\n\u003cli\u003eSomiya, M., Y. Yoshioka, and T. Ochiya, \u003cem\u003eBiocompatibility of highly purified bovine milk-derived extracellular vesicles.\u003c/em\u003e J Extracell Vesicles, 2018. \u003cstrong\u003e7\u003c/strong\u003e(1): p. 1440132.\u003c/li\u003e\n\u003cli\u003eWu, P., et al., \u003cem\u003eExtracellular vesicles: A bright star of nanomedicine.\u003c/em\u003e Biomaterials, 2021. \u003cstrong\u003e269\u003c/strong\u003e: p. 120467.\u003c/li\u003e\n\u003cli\u003eZhu, L.P., et al., \u003cem\u003eHypoxia-elicited mesenchymal stem cell-derived exosomes facilitates cardiac repair through miR-125b-mediated prevention of cell death in myocardial infarction.\u003c/em\u003e Theranostics, 2018. \u003cstrong\u003e8\u003c/strong\u003e(22): p. 6163-6177.\u003c/li\u003e\n\u003cli\u003eTian, J., et al., \u003cem\u003eEngineered Exosome for Drug Delivery: Recent Development and Clinical Applications.\u003c/em\u003e Int J Nanomedicine, 2023. \u003cstrong\u003e18\u003c/strong\u003e: p. 7923-7940.\u003c/li\u003e\n\u003cli\u003eChen, Z., et al., \u003cem\u003eEncapsulation and assessment of therapeutic cargo in engineered exosomes: a systematic review.\u003c/em\u003e J Nanobiotechnology, 2024. \u003cstrong\u003e22\u003c/strong\u003e(1): p. 18.\u003c/li\u003e\n\u003cli\u003eRahbarghazi, R., et al., \u003cem\u003eTumor-derived extracellular vesicles: reliable tools for Cancer diagnosis and clinical applications.\u003c/em\u003e Cell Commun Signal, 2019. \u003cstrong\u003e17\u003c/strong\u003e(1): p. 73.\u003c/li\u003e\n\u003cli\u003eChang, W.H., R.A. Cerione, and M.A. Antonyak, \u003cem\u003eExtracellular Vesicles and Their Roles in Cancer Progression.\u003c/em\u003e Methods Mol Biol, 2021. \u003cstrong\u003e2174\u003c/strong\u003e: p. 143-170.\u003c/li\u003e\n\u003cli\u003eHou, Y., J. Wang, and J. Wang, \u003cem\u003eEngineered biomaterial delivery strategies are used to reduce cardiotoxicity in osteosarcoma.\u003c/em\u003e Front Pharmacol, 2023. \u003cstrong\u003e14\u003c/strong\u003e: p. 1284406.\u003c/li\u003e\n\u003cli\u003eLiu, X., C. Xiao, and K. Xiao, \u003cem\u003eEngineered extracellular vesicles-like biomimetic nanoparticles as an emerging platform for targeted cancer therapy.\u003c/em\u003e J Nanobiotechnology, 2023. \u003cstrong\u003e21\u003c/strong\u003e(1): p. 287.\u003c/li\u003e\n\u003cli\u003eStridfeldt, F., et al., \u003cem\u003eAnalyses of single extracellular vesicles from non-small lung cancer cells to reveal effects of epidermal growth factor receptor inhibitor treatments.\u003c/em\u003e Talanta, 2023. \u003cstrong\u003e259\u003c/strong\u003e: p. 124553.\u003c/li\u003e\n\u003cli\u003eKooijmans, S.A.A., et al., \u003cem\u003ePEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time.\u003c/em\u003e J Control Release, 2016. \u003cstrong\u003e224\u003c/strong\u003e: p. 77-85.\u003c/li\u003e\n\u003cli\u003eLi, Y., et al., \u003cem\u003eA33 antibody-functionalized exosomes for targeted delivery of doxorubicin against colorectal cancer.\u003c/em\u003e Nanomedicine, 2018. \u003cstrong\u003e14\u003c/strong\u003e(7): p. 1973-1985.\u003c/li\u003e\n\u003cli\u003eShi, X., et al., \u003cem\u003eGenetically Engineered Cell-Derived Nanoparticles for Targeted Breast Cancer Immunotherapy.\u003c/em\u003e Mol Ther, 2020. \u003cstrong\u003e28\u003c/strong\u003e(2): p. 536-547.\u003c/li\u003e\n\u003cli\u003eCheng, Q., et al., \u003cem\u003eReprogramming Exosomes as Nanoscale Controllers of Cellular Immunity.\u003c/em\u003e J Am Chem Soc, 2018. \u003cstrong\u003e140\u003c/strong\u003e(48): p. 16413-16417.\u003c/li\u003e\n\u003cli\u003eChao, M.P., I.L. Weissman, and R. Majeti, \u003cem\u003eThe CD47-SIRPalpha pathway in cancer immune evasion and potential therapeutic implications.\u003c/em\u003e Curr Opin Immunol, 2012. \u003cstrong\u003e24\u003c/strong\u003e(2): p. 225-32.\u003c/li\u003e\n\u003cli\u003eLin, W.W., et al., \u003cem\u003eEnhancement Effect of a Variable Topology of a Membrane-Tethered Anti-Poly(ethylene glycol) Antibody on the Sensitivity for Quantifying PEG and PEGylated Molecules.\u003c/em\u003e Anal Chem, 2017. \u003cstrong\u003e89\u003c/strong\u003e(11): p. 6082-6090.\u003c/li\u003e\n\u003cli\u003eLiang, G., et al., \u003cem\u003eEngineered exosome-mediated delivery of functionally active miR-26a and its enhanced suppression effect in HepG2 cells.\u003c/em\u003e Int J Nanomedicine, 2018. \u003cstrong\u003e13\u003c/strong\u003e: p. 585-599.\u003c/li\u003e\n\u003cli\u003eOhno, S., et al., \u003cem\u003eSystemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells.\u003c/em\u003e Mol Ther, 2013. \u003cstrong\u003e21\u003c/strong\u003e(1): p. 185-91.\u003c/li\u003e\n\u003cli\u003eCheng, Q., X. Shi, and Y. Zhang, \u003cem\u003eReprogramming Exosomes for Immunotherapy.\u003c/em\u003e Methods Mol Biol, 2020. \u003cstrong\u003e2097\u003c/strong\u003e: p. 197-209.\u003c/li\u003e\n\u003cli\u003eFerreira, D., J.N. Moreira, and L.R. Rodrigues, \u003cem\u003eNew advances in exosome-based targeted drug delivery systems.\u003c/em\u003e Crit Rev Oncol Hematol, 2022. \u003cstrong\u003e172\u003c/strong\u003e: p. 103628.\u003c/li\u003e\n\u003cli\u003eZheng, W., et al., \u003cem\u003eInhalable CAR-T cell-derived exosomes as paclitaxel carriers for treating lung cancer.\u003c/em\u003e J Transl Med, 2023. \u003cstrong\u003e21\u003c/strong\u003e(1): p. 383.\u003c/li\u003e\n\u003cli\u003eLee, H.H., et al., \u003cem\u003eTherapeutic effiacy of T cells expressing chimeric antigen receptor derived from a mesothelin-specific scFv in orthotopic human pancreatic cancer animal models.\u003c/em\u003e Neoplasia, 2022. \u003cstrong\u003e24\u003c/strong\u003e(2): p. 98-108.\u003c/li\u003e\n\u003cli\u003eWang, Y., et al., \u003cem\u003eChemokine Receptor CCR2b Enhanced Anti-tumor Function of Chimeric Antigen Receptor T Cells Targeting Mesothelin in a Non-small-cell Lung Carcinoma Model.\u003c/em\u003e Front Immunol, 2021. \u003cstrong\u003e12\u003c/strong\u003e: p. 628906.\u003c/li\u003e\n\u003cli\u003eWang, P., et al., \u003cem\u003eExosomes from M1-Polarized Macrophages Enhance Paclitaxel Antitumor Activity by Activating Macrophages-Mediated Inflammation.\u003c/em\u003e Theranostics, 2019. \u003cstrong\u003e9\u003c/strong\u003e(6): p. 1714-1727.\u003c/li\u003e\n\u003cli\u003eRezakhani, L., et al., \u003cem\u003eExosomes: special nano-therapeutic carrier for cancers, overview on anticancer drugs.\u003c/em\u003e Med Oncol, 2022. \u003cstrong\u003e40\u003c/strong\u003e(1): p. 31.\u003c/li\u003e\n\u003cli\u003eLennaard, A.J., et al., \u003cem\u003eOptimised Electroporation for Loading of Extracellular Vesicles with Doxorubicin.\u003c/em\u003e Pharmaceutics, 2021. \u003cstrong\u003e14\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eSutaria, D.S., et al., \u003cem\u003eAchieving the Promise of Therapeutic Extracellular Vesicles: The Devil is in Details of Therapeutic Loading.\u003c/em\u003e Pharm Res, 2017. \u003cstrong\u003e34\u003c/strong\u003e(5): p. 1053-1066.\u003c/li\u003e\n\u003cli\u003eSchindler, C., et al., \u003cem\u003eExosomal delivery of doxorubicin enables rapid cell entry and enhanced in vitro potency.\u003c/em\u003e PLoS One, 2019. \u003cstrong\u003e14\u003c/strong\u003e(3): p. e0214545.\u003c/li\u003e\n\u003cli\u003eUslu, D., et al., \u003cem\u003eEffect of platelet exosomes loaded with doxorubicin as a targeted therapy on triple-negative breast cancer cells.\u003c/em\u003e Mol Divers, 2022.\u003c/li\u003e\n\u003cli\u003eZhou, G., et al., \u003cem\u003eExosome Mediated Cytosolic Cisplatin Delivery Through Clathrin-Independent Endocytosis and Enhanced Anti-cancer Effect via Avoiding Endosome Trapping in Cisplatin-Resistant Ovarian Cancer.\u003c/em\u003e Front Med (Lausanne), 2022. \u003cstrong\u003e9\u003c/strong\u003e: p. 810761.\u003c/li\u003e\n\u003cli\u003eLimoni, S.K., et al., \u003cem\u003eEngineered Exosomes for Targeted Transfer of siRNA to HER2 Positive Breast Cancer Cells.\u003c/em\u003e Appl Biochem Biotechnol, 2019. \u003cstrong\u003e187\u003c/strong\u003e(1): p. 352-364.\u003c/li\u003e\n\u003cli\u003eTian, Y., et al., \u003cem\u003eA doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy.\u003c/em\u003e Biomaterials, 2014. \u003cstrong\u003e35\u003c/strong\u003e(7): p. 2383-90.\u003c/li\u003e\n\u003cli\u003eLiang, G., et al., \u003cem\u003eEngineered exosomes for targeted co-delivery of miR-21 inhibitor and chemotherapeutics to reverse drug resistance in colon cancer.\u003c/em\u003e J Nanobiotechnology, 2020. \u003cstrong\u003e18\u003c/strong\u003e(1): p. 10.\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":"cancer-nanotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cano","sideBox":"Learn more about [Cancer Nanotechnology](https://cancer-nano.biomedcentral.com/)","snPcode":"12645","submissionUrl":"https://submission.nature.com/new-submission/12645/3","title":"Cancer Nanotechnology","twitterHandle":"@CancerNanotech","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Extracellular Vesicles, Oxaliplatin Delivery and EGFR+ Colorectal Cancer","lastPublishedDoi":"10.21203/rs.3.rs-4291698/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4291698/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e To investigate the ability of extracellular vesicles (EVs) to deliver oxaliplatin to epidermal growth factor receptor (EGFR\u003csup\u003e+\u003c/sup\u003e) colorectal cancer cells and increase oxaliplatin’s cytotoxicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod: \u003c/strong\u003eOxaliplatin was passively loaded into a stable cell line expressing cetuximab in membranes. EVs were collected and characterized for size, and their ability to target EGFR\u003csup\u003e+\u003c/sup\u003e cells was tested. Cytotoxicity experiments were performed, and a xenograft cancer animal model was used to confirm the specific accumulation of oxaliplatin-loaded EVs with cetuximab-expressing membranes in EGFR\u003csup\u003e+\u003c/sup\u003e cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eEVs with cetuximab-expressing membranes were successfully produced and used to encapsulate oxaliplatin, resulting in consistently sized oxaliplatin-loaded EVs with cetuximab-expressing membranes. The oxaliplatin-loaded EVs with cetuximab-expressing membranes were specifically internalized by EGFR\u003csup\u003e+\u003c/sup\u003e cells, leading to significant cytotoxic effects on these cells. In the animal model, the oxaliplatin-loaded EVs with cetuximab-expressing membranes accumulated specifically in EGFR\u003csup\u003e+\u003c/sup\u003e cells and significantly enhanced oxaliplatin’s therapeutic efficacy against EGFR\u003csup\u003e+\u003c/sup\u003e cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eEVs with membrane-expressed bioactive molecules are a promising strategy for delivering therapeutic agents to EGFR\u003csup\u003e+\u003c/sup\u003e colorectal cancer cells.\u003c/p\u003e","manuscriptTitle":"Maximizing Oxaliplatin's Impact on EGFR+ Colorectal Cancer Through Targeted Extracellular Vesicles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 19:27:42","doi":"10.21203/rs.3.rs-4291698/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-19T23:17:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-19T15:52:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-11T13:25:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69136424285236566244397124212829990430","date":"2024-06-10T08:55:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277065078329563444087308619247991748390","date":"2024-06-09T23:56:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-16T00:14:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200727908510949847303553300428583330208","date":"2024-05-06T00:16:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-02T23:16:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-22T10:15:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-22T10:15:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Nanotechnology","date":"2024-04-19T07:57:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-nanotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cano","sideBox":"Learn more about [Cancer Nanotechnology](https://cancer-nano.biomedcentral.com/)","snPcode":"12645","submissionUrl":"https://submission.nature.com/new-submission/12645/3","title":"Cancer Nanotechnology","twitterHandle":"@CancerNanotech","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5cde6961-853f-4495-a512-985d39f1781d","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-22T19:44:08+00:00","versionOfRecord":{"articleIdentity":"rs-4291698","link":"https://doi.org/10.1186/s12645-024-00284-0","journal":{"identity":"cancer-nanotechnology","isVorOnly":false,"title":"Cancer Nanotechnology"},"publishedOn":"2024-08-17 15:58:17","publishedOnDateReadable":"August 17th, 2024"},"versionCreatedAt":"2024-04-25 19:27:42","video":"","vorDoi":"10.1186/s12645-024-00284-0","vorDoiUrl":"https://doi.org/10.1186/s12645-024-00284-0","workflowStages":[]},"version":"v1","identity":"rs-4291698","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4291698","identity":"rs-4291698","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}