Preparation of microalgae-derived exosomes drug delivery system loaded with oridonin and evaluation of anti-colorectal cancer effect

Preparation of microalgae-derived exosomes drug delivery system loaded with oridonin and evaluation of anti-colorectal cancer effect | 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 Preparation of microalgae-derived exosomes drug delivery system loaded with oridonin and evaluation of anti-colorectal cancer effect Baiyan Wang, Aifang Li, Shuxuan Li, Wei Chen, Yalan Li, Lei Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8418021/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Some Chinese medicine monomers are limited in clinical application due to poor targeting and in vivo stability, strong hydrophobicity, low bioavailability, short half-life, and systemic toxicity within the therapeutic dose range, etc. Combining these monomers with nanotechnology-based drug delivery systems can further improve the targeting and clinical efficacy of these drugs. Thus, this study, constructed a drug delivery vector targeting colorectal cancer (CRC), i.e. cRGD-DEXO with cRGD-modified exosomes of Dunaliella salina (DEXO), and their effects of targeting and anti-CRC were investigated at the cellular level in vitro and in animal model in vivo . The results verified that the anti-CRC effect is cRGD-DEXO/ORI>DEXO/ORI>ORI in vitro . In vivo studies showed that anti-tumor effect of CRC in nude mice was cRGD-DEXO/ORI>ORI. In addition, compared with non-cRGD-modified DEXO, cRGD-modified DEXO showed stronger targeting to HCT-116 cells. Furthermore, cRGD-DEXO/ORI showed significantly higher accumulation in nude mouse tumor tissues than non-cRGD-modified DEXO/ORI and free ORI, directly enhancing its in vivo anti-tumor activity by concentrating ORI at tumor sites. Summarily, our study proved that we have successfully constructed a drug delivery system (i.e. cRGD-DEXO/ORI) that targets CRC, and it exhabits a better anti-CRC activity both in vivo and in vitro . It provided a promising targeted delivery strategy for hydrophobic Chinese medicine monomers like ORI, laying a solid experimental foundation for their potential clinical translation and offering new insights into the application of nanotechnology in optimizing the therapeutic efficacy of traditional Chinese medicine against malignant tumors. Colorectal cancer Drug delivery system Dunaliella salina exosomes Oridonin Targeting delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Epidemiological data show an increasing of colorectal cancer (CRC) year by year. Although significant advancements in chemotherapy, radiotherapy and targeted therapy, the prognosis of advanced CRC remains poor, with a persistently low 5-year survival rate [1] . Traditional Chinese medicine (TCM) exhibits multi-target and multi-pathway properties in diseases treatment, which is an important means of cancer management in China, especially for advanced malignancies. Numerous plant-based compounds have been shown to have benefits for disease and health. With the rapid development of drug mechanism separation technology and evaluation methods, an increasing number of active ingredients of TCM have been identified and verified for anti-tumor efficacy [2] . For instance, studies have shown that oridonin (ORI) [3] , berberine (BBR) [4] and baicalin (BAI) [5] all had proliferation-inhibiting effects on CRC cell. However, their clinical application is always hindered by poor stability, inadequate targeting, short half-life and low bioavailability etc. The rapid development of nanotechnology has successfully enabled nanoparticles to be widely applied as drug delivery systems cancer therapy. The nanization of TCM components has shown great advantages in recent years. Exosome (EXO), natural extracellular vesicle with a diameter of 30~150 nm secreted by various cells, are composed of lipids, proteins, and RNAs [6] . As key mediators of intercellular communication, they play an important role in transmitting genetic and biochemical information between cells [7] , emerging as promising drug delivery carriers [8] . Their advantages include: (1) excellent biocompatibility, low immunogenicity and minimal long-term safety concerns [9] ; (2) membrane proteins, lipids and polysaccharides conferring inherent homing properties and natural sites for artificial modification [10] ; and (3) efficient penetration of biological barriers [11] . However, the high cost of EXO isolation from animal cells limits their scalability. Fortunately, marine algae produce diverse bioactive metabolites with reported anticancer properties, offering alternative sources for exosome production [12] . Dunaliella salina ( Ds ) , a unicellular eukaryotic green algae thriving in bracken water, possesses remarkable natural advantages for genetic engineering and dpractical applications. Firstly, it is non-toxic, nutrient-rich, and enhances immune function and resistance as a natural health product [13] . Secondly, its lack of a cell wall (natural protoplast state) facilitates foreign genes transfection, holding promise for genetic engineering [14] . Compared to exosomes from other sources, Ds exosomes (DEXO) offer high modifiability, stability transformation, low endogenous toxicity, and scalable production potential. Furthermore, DEXO include intrinsic anticancer agents such as polysaccharides and β-carotene [15] , which can play a synergistic role as drug carriers. In this study, cRGD was conjugated to DEXO membranes via chemical click reaction, and ORI was loaded into cRGD-DEXO using electroporation to construct an targeted drug delivery system (cRGD-DEXO/ORI) for CRC. Its anti-CRC efficacy was evaluated both in vivo and in vitro . This research holds dual significance: (1) it pioneers the application of DEXO as drug delivery vectors, providing theoretical and methodological insights for further carrier development; (2) it enhances the anti-CRC efficacy of ORI, offering strategies for clinical application of ORI. 2. Results 2.1 Effects of ORI, BBR and BAI on the activity of HCT-116 cells As shown in Figure 1 A1, MTT results showed that after ORI was treated on HCT-116 cells for 24 h, ORI exhibited dose-dependent antiproliferative effects across concentrations of 6.25, 12.5, 25, 50, 100, and 200 µg/mL relative to the negative control group. All tested concentrations exerted statistically significant inhibitory effects on cell proliferation( P <0.0001). A clear dose-dependent decrease in cell survival rate was observed: specifically, survival rate declined significantly as ORI concentrations increased from 3.125 to 25 µg/mL, dropping to approximately 3% at 25 µg/mL and remaining unchanged with further increases in ORI concentration (up to 200 µg/mL). For 48h ORI treatment, HCT-116 cells displayed a similar dose-dependent trend in cell survival rate as the 24h group. At 12.5 µg/mL ORI, cell survival rate was only 9.19%, and no further significant decrease was noted with higher concentrations. Furthermore, the antiproliferative effects of ORI at concentrations of 12.5, 25, 50, 100, and 200 µg/mL after 48 h were not significantly different from those observed at 24 h. Treatment of HCT-116 cells with BBR for 24 h showed no significant inhibitory effect at concentrations less than 50 µg/mL (vs. negative control). Only at concentrations greater than or equal to 50 µg/mL did BBR exert inhibition, with cell viability remaining 76% at 200 µg/mL (Figure 1A3). After 48 h of BBR treatment, strong proliferation inhibition was observed at concentrations greater than or equal 6.25 µg/mL, cell viability dropped to 37% at 200 µg/mL (Figure 1A4). For BAI: After 24 h, cell viability was unaffected at concentrations less than 50 µg/mL (vs. negative control). Viability began to decline at 50 µg/mL, remaining 72% at 200 µg/mL. After 48 h, BAI had no inhibitory effect at concentrations less than 50 µg/mL. However, unlike the 24 h group, cell viability decreased to 43% at 200 µg/mL (Figure 1A6). 2.2 Effect of ORI on the morphology of HCT-116 and LO2 cells After HCT-116 cells were treated with ORI at concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125 µg/mL for 24 h, significant morphological changes were observed. With increasing ORI concentration, cells gradually transformed from a spindle-shaped to a rounded morphology. Notably, most cell exhibited membrane budding, resembling apoptotic bodies (Figure 1, A7). To verify whether the ORI concentration that inhibits HCT-116 cells proliferation by 70% (IC₇₀) induces cytotoxicity on normal liver LO2 cells, we exposed LO2 cells to this IC70 concentration (Figure 1, A8) and the results demonstrated that compared with the untreated LO2 cells control group, LO2 cells treated with 6.25 µg/mL showed a long spindle-shape morphology, maintained a healthy growth state with uniform distribution and high density. 2.3 Characterization and identification of DEXO and cRGD-DEXO/ORI TEM revealed that DEXO exhibited a "saucer-like" structure with intact morphology, consistent with the established morphological features of typical exosome (Figure 2, B1). In contrast, cRGD-DEXO/ORI displayed a "spherical" structure and maintained structural completeness (Figure 2, B2). Zeta potentials and particle size distributions of DEXO and cRGD-DEXO/ORI were measured by a DLS analyzer. Results showed that both DEXO (Figure 2, B3) and cRGD-DEXO/ORI (Figure 2, B4) exhibited zeta potentials ranging from -30~-50 mv, with cRGD-DEXO/ORI showing a modest reduction in surface charge compared to DEXO. Regarding particle size, DEXO was predominantly distributed within the range of 90~140 nm (Figure 2, B5), whereas cRGD-DEXO/ORI displayed a slight increase in size, with a broader distribution spanning 92~295 nm and a main peak concentrated at 164~255 nm (Figure 2, B6). Both samples exhibited PDI. Collectively, All these findings confirm the successful isolation of DEXO and demonstrate that both DEXO and cRGD-EXO/ORI conform to the established physicochemical characteristics of exosomes. 2.4 Construction and optimization of cRGD-DEXO/ORI As shown in Figure 3 C1, blue fluorescence (DAPI staining) indicates the nuclei of HCT-116 cells, while green fluorescence (PKH67 labeling) corresponds to DEXO. The merged image demonstrates colocalization of DEXO and HCT-116 cells. CLSM revealed that PKH67-labeled DEXO and cRGD-DEXO localized to both the cell membrane and cytoplasm of HCT-116 cells. Notably, compared with unmodified DEXO, cRGD-DEXO exhibited significantly stronger fluorescence intensity in their cell membrane and cytoplasm. These results indicate that cRGD modification enhances the targeting ability of DEXO to HCT-116 cells and promotes their internalization. Figure 3 C2 illustrates the time-dependent uptake of cRGD-DEXO by HCT-116 cells at different time points. CLSM showed that at 12h, numerous green fluorescent dots (PKH67-labeled cRGD-DEXO) formed ring-like distributions around the cell membrane and nucleus. With extended incubation, green fluorescence signals also appeared within the nucleus. This observation was attributed to the tumor-penetrating properties of cRGD, potentially via a "hole" effect. Collectively, these results indicated that cRGD-DEXO not only targets the cell membrane of HCT-116 cells but also undergoes internalization, with intracellular accumulation increasing over time. In addition, as shown in Figure 3 C3, the binding ability of DEXO was optimal at a DEXO to cRGD ratio of 5:1. Therefore, the cellular uptake of cRGD-DEXO by HCT-116 cells exhibited a time-dependent increase. 2.5 Establishment of ORI standard curve As shown in Figure 3 C4, analysis the relationship between ORI concentration and absorbance at 238 nm yielded a standard curve equation of y=0.003x+3.34 (R2=0.9837), spanning a linear range of 0.78125~25 µg/mL. These results demonstrate a strong linear correlation between ORI concentration and absorbance at 238 nm, which is sufficient to meet the requirements for quantitative analysis and subsequent experiments procedures. 2.6 Determination of loading rate and encapsulation rate of cRGD-DEXO and ORI As shown in Figure 3 C5, the loading rate and encapsulation rate of ORI were quantified using a microplate reader at different ratios of cRGD-DEXO and ORI. The results demonstrated that increasing ORI concentration led to a downward trend in encapsulation rate, while the loading rate stabilized. Conversely, increasing cRGD-DEXO concentration resulted in an upward trend in the encapsulation rate accompanied by a slight decrease in loading rate. At an ORI:cRGD-DEXO ratio of 1:1, the loading rate was 22.3% and encapsulation rate was 29%, representing an optimal balanced that maximized effective utilization of both components. Therefore, the 1:1 ORI: cRGD-DEXO of ratio was selected for subsequent experiments. 2.7 cRGD-DEXO/ORI safety range evaluation and its effect on HCT-116 cells To evaluate the biocompatibility of cRGD-DEXO/ORI, LO2 cell line was used as an in vitro model to assess its safe concentration range. LO2 cells were treated with different concentrations cRGD-DEXO/ORI, and the effect of cRGD-DEXO /ORI on LO2 cells viability was detected by MTT assay. As shown in Figure 3 C6, after 24 h co-incubation of cRGD-DEXO/ORI with the cells, no inhibitory effect on LO2 cells proliferation was observed with cell viability remaining above 80%. However, when the concentration of cRGD-DEXO/ORI reached 500 µg/mL, cell viability dropped below 80%.To evaluate the antiproliferative efficacy of cRGD-DEXO/ORI in vitro , HCT-116 cells were treated with different formulations for 24 h, and cell viability was analyzed. As shown in Figure 4 D1, the cell survival rate in DEXO/ORI group was significantly lower than that in ORI group ( P <0.001). Furthermore, the cell proliferation inhibition ability of cRGD-DEXO/ORI group was stronger than that of DEXO/ORI group ( P <0.01). However, no significant difference was observed between the cRGD-DEXO/ORI and 5-FU groups. In addition, DEXO group showed no inhibitory effect on proliferation of HCT-116 cells, with cell survival rate remaining as high as 97%. Notably, ORI encapsulated in DEXO (i.e., DEXO/ORI) exerted a potent inhibitory effect on cell proliferation: compared with ORI group, cell survival rate in DEXO/ORI group was reduced by approximately 1.6-fold. Following modification of DEXO/ORI with cRGD, cell survival rate was reduced by 1.85-fold compared with DEXO/ORI group. Overall, cRGD-DEXO/ORI intervention resulted in a significant 5-fold reduction in HCT-116 cell viability relative to the negative control group. In conclusion, cRGD-DEXO/ORI exhibited favorable in vitro inhibitory activity against CRC cells proliferation, and ORI encapsulated in cRGD-modified DEXO showed enhanced antiproliferative efficacy compared to ORI. 2.8 Effects of cRGD-DEXO/ORI on apoptosis of HCT-116 cells AO can penetrate complete cell membranes and intercalate into nuclear DNA, exhibiting bright green fluorescence. In contrast, EB can only permeates cells with compromised membranes and intercalates into nuclear DNA, emitting orange fluorescence. Under the fluorescence microscope, four different cell morphology can be clearly distinguished: viable cells exhibit green and maintains a normal structure. The chromatin of early apoptotic cells also showed green fluorescence but with condensed or beaded fragmented chromatin. In contrast, non-apoptotic dead cells show orange-stained chromatin that maintains normal structure, whereas the chromatin of late apoptotic cells present orange-red chromatin with pronounced condensation or fragmentation. These morphological differences provide an intuitive and accurate method for assessing cellular status. As shown in Figure 4 D2, D3 and D4, the late apoptosis rate of HCT-116 cells in the cRGD-DEXO/ORI group was statistically significantly increased compared with the negative control group. The late apoptosis rates accounted for 21%, 63%, 75% and 64% of total cells in ORI, DEXO/ORI, cRGD-DEXO/ORI and 5-FU groups, respectively. In contrast, cell in the negative control group and DEXO groups exhibited normal growth, with dead cells comprising only 9% and 11% of total cell population, respectively. In conclusion, cRGD-DEXO/ORI markedly enhances the apoptosis rate of HCT-116 cells, primarily inducing late apoptotic phenotypes, whereas DEXO alone has no inhibitory effect on HCT-116 cells growth. 2.9 Effects of cRGD-DEXO/ORI on HCT-116 cells migration Figure 4 D5 and D6 demonstrate the effects of different drugs groups on HCT-116 cell migration after 24 h. The results showed that the relative migration area in the DEXO/ORI, cRGD-DEXO/ORI and 5-FU groups was significantly lower than that in negative control group. Compared with ORI alone, the inhibition effect of cRGD-DEXO coated ORI on HCT-116 cells migration was approximately twice as potent. Notably, the relative migration area in cRGD-DEXO/ORI group was the smallest, accounting for roughly 1/3 of that in negative control group. In conclusion, the DEXO/ORI, cRGD-DEXO/ORI and 5-FU groups all exerted inhibitory effects on HCT-116 cell migration, with the cRGD-DEXO/ORI group exhibiting the most pronounced inhibitory effect. 2.10 In vivo anti-tumor evaluation of subcutaneous tumors in nude mice The effects of cRGD-DEXO/ORI on tumor growth inhibition and body weight were evaluated in BALB/c nude mice. As shown in Figure 5 E1, compared with the model group, the ORI, cRGD-DEXO/ORI and 5-FU groups had different degrees of inhibition on CRC growth. Notably, the growth inhibition effect of cRGD-DEXO/ORI on CRC was more obvious than that of ORI alone or 5-FU groups. On day 18, the tumor volumes in model, ORI and 5-FU groups were 2.59-, 1.91-, and 1.38-fold those in cRGD-DEXO/ORI group, respectively. In addition, as shown in Figure 5 E2, the different drug treatments exerted no significant effect on the body weight of nude mice. Collectively, these results indicate that ORI exhibits potent anti-CRC activity, and this activity is significantly enhanced when ORI is encapsulated in cRGD-DEXO. As shown in Figure 5 E3 and E4, the change curves of tumor fluorescence intensity changes over time in nude mice with CRC xenografts treated with different drugs. The results demonstrated that tumor fluorescence intensity was enhanced to varying degrees over time. Meanwhile, on day 18, ORI ( P <0.05), cRGD-DEXO/ORI ( P <0.01), and 5-FU ( P <0.01) groups had weaker fluorescence intensity than the model group, with reductions of 1.27-, 1.42-, and 1.30-fold, respectively. In addition, ORI group fluorescence was 0.89-fold (vs cRGD-DEXO/ORI, P <0.05) and 0.98-fold times (vs 5-FU, P <0.05) that of latter two groups, with no significant difference between the 5-FU and the cRGD-DEXO/ORI groups. In view of this, cRGD-DEXO/ORI exhibits a certain capacity to inhibit CRC growth. Meanwhile, as shown in Figure 5 E5 and E6, tumor volume and tumor inhibition rate analyses revealed that the anti-CRC activity of cRGD-DEXO/ORI was superior to that of ORI group ( P <0.05). Compared with ORI group, 5-FU exerted a more pronounced inhibitory effect on CRC growth ( P 0.05). The tumor inhibition rates in the ORI, cRGD-DEXO/ORI and 5-FU groups were 26%, 61% and 47%, respectively. 3. Materials and methods 3.1 Culture of HCT-116 cells Human CRC HCT-116 cells were supplied by Shanghai Fuheng Material Technology Co., Ltd. (Shanghai, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37℃ under a humidified atmosphere containing 5% CO 2 , with medium refreshed every 48-72 h to ensure optimal nutrient supply. Cells were passaged at 90% confluency: spent medium was aspirated, cells rinsed three times with pre-warmed PBS (pH 7.4), then 2 mL 0.25% trypsin-EDTA added and incubated at 37°C for 2~3 min until rounded. Digestion was terminated with 3 mL complete DMEM (10% FBS), and cells were pipetted to a single-cell suspension, transferred to a 15 mL tube, centrifuged at 1000 rpm for 5 min. Supernatant was aspirated, pellet resuspended in fresh medium, seeded 1:3 into new T-75 flasks, and returned to the incubator for expansion. 3.2 Comparison of antitumor activity of ORI, BBR and BAI ORI, BBR and BAI were purchased from Chengdu Master Biotechnology Co., Ltd., and were prepared at 7 concentration gradients (200, 100, 50, 25, 12.5, 6.25, 3.125 µg/mL, alongside a negative control group and a blank control group. The required volume of drug working solution was calculated, and solution were diluted to target concentrations using DMEM containing 10% FBS. 96-well plates were removed from the CO 2 incubator, original medium were discarded, and drug-containing media at gradient concentrations were added to wells (200 µL per well). Each concentration was tested in 6 replicate wells, and plates were labeled with date, time and dosage. Incubation was continued in the CO 2 incubator for 24 and 48h, respectively. After incubation, medium was aspirated, and 200 µL of pre-prepared 5 mg/mL MTT solution was added to each well (protected from light). Following 4 h of incubation, MTT solution was discarded, and 150 µL DMSO was added to dissolve blue-violet formazan crystals (produced by succinic dehydrogenase-mediated reduction of MTT in viable cells). Plates were shaken for 10 min to ensure complete crystal dissolution. Absorbance (OD) at 490 nm was measured using a microplate reader. Cell survival rate=(experimental group OD-blank group OD)/(control group OD-blank group OD)×100%. The experiment was repeated three times. 3.3 Effects of ORI on the morphology of HCT-116 and LO2 cells HCT-116 cells culture and plate seeding were performed as described above. ORI was added to HCT-116 cells at concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125 µg/mL. After 24 h of treatment, cells were observed and photographed under an inverted microscope. Culture of normal hepatocyte LO2 (obtained from the First Clinical School of Medicine) and plate seeding were performed as those for HCT-116 cells. When cell density reached 70%, 200µL of 6.25 µg/mL ORI was added to each well. After 24 h, the morphology of LO2 cells was observed under an inverted fluorescence microscope. 3.4 Culture of Ds cells To prepare the medium, 950 mL of distilled water was first measured, and the following reagents were sequentially dissolved in it: NaNO 3 (75 g/L), NaH 2 PO 4 · H 2 O (5 g/L), Na 2 SiO 3 ·9H 2 O (30 g/L), CuSO 4 ·5H 2 O (9.8 g/L), Na 2 MoO 4 ·2H 2 O (6.3 g/L) and ZnSO 4 ·7H₂O(22.0 g/L), CoCl 2 ·6H 2 O (10.0 g/L), MnCl 2 ·4H 2 O (180.0 g/L), Vitamin B12 (1 mg/mL), Biotin (0.1 mg/mL). The solution was then quantified to 1L with distilled water, and the pH was adjusted to 8 using NaOH before autoclaving for sterilization. Ds cells were retrieved from cryovials and quickly thawed in a 37°C water bath and then carefully and slowly added to the PKS liquid medium using a sterile pipette. The cells were cultured overnight in an illuminated incubator at 26°C with a 14:10 light/dark cycle. The following day, Ds cell concentration was quantified using a visible-light spectrophotometer, and 50 μL cell suspension (1×10 6 cells /mL) was plated onto PKS solid medium by sterile pipette. Cultivation was continued under the same conditions (26°C, 14:10 light/dark cycle) until distinct monoclonal colonies had formed. Individual colonies were carefully selected and transferred to fresh PKS solid medium, then scaled up to 1 L PKS liquid medium for further cultivation. After incubation 3 d, when the cell concentration reached 1×10 6 cells/mL, cell expansion was performed. Finally, the 2nd to 4th generation of Ds cell cultures were selected as high-quality starting material for extracting exosome extraction. 3.5 Extraction and characterization of DEXO 3.5.1 Extraction of DEXO After 7 d of culture, the Ds cell culture medium was collected for DEXO extraction by ultracentrifugation, following these steps: First, the medium was centrifuged at 4°C, 2,000× g for 10 min to remove intact algal cells; the resulting supernatant was then centrifuged at 4°C, 10,000× g for 30 min to remove cell debris. Next, the clarified supernatant was ultracentrifuged at 4°C, 11,8000× g for 90 min. After discarding the supernatant, the exosomes-enriched pellet adhering to the ultracentrifuge tube wall was resuspended in PBS, filtered with 0.22 µm filter membrane, and ultracentrifuged again at 4°C, 11,8000× g for 90 min. The final supernatant was resuspended in a small amount of PBS solution to obtain DEXO. Protein concentration of DEXO was quantified using the BCA method. If the concentration was too low, additional ultracentrifugation was performed for further enrichment. Aliquots were stored at -80°C, with a storage period of 6 months, and repeated free-thaw cycles were avoided. 3.5.2 Characterization of DEXO For transmission electron microscopy (TEM) analysis, parafilm was cut into sections, and a copper mesh was placed onto the droplet with tweezers for 5 min incubation. Excess sample was blotted with filter paper, and 10 µL 3% uranyl acetate was dropped on a fresh parafilm section. The copper mesh was transferred onto the staining solution, excess stain was blotted, and the mesh was air-dried. Finally, the copper mesh was imaged via TEM with micrographs captured for documentation. Open the machine according to the instructions, and use a 1mL pipette to move the sample into the cuvette. The sample volume is equal to the scale on the cuvette. After that, cuvette was inserted into the instrument’s sample compartment and measurements were initiated. If the polydispersity index (PDI) exceeded the acceptable threshold, sample concentration may be too high or agglomeration. Appropriate dilution or ultrasonic treatment was performed as necessary, the measurement is repeated three times, with experimental data recorded and corresponding graphs generated for analysis. 3.6 Construction and optimization of cRGD-DEXO DBCO is a reactive cyclooctyne used for strain-promoted azides-alkyne cycloaddition, which can react with substances containing primary or secondary amino groups without copper. 1mg/mL DBCO solution was added to 1mg/mL DEXO solution, and the mixture was incubated on a mixer at 4℃ and pH 7.4 for 4 h. After incubation, the mixture was centrifuged at 16,4000× g for 90 min, the supernatant was discarded, and the pellet then washed and resuspended in PBS to obtain DBCO-DEXO. The protein concentration was measured using a BCA assay. For optimizing the ratio of DBCO-DEXO to cRGD, mass ratios of 1:1, 1:2, 1:5, 2:1 and 5:1 (μg:μg) were set, and the reaction was incubated on a rotary mixer at 4℃ and pH 7.4 for 12 h. The mixture was then centrifuged at 16,4000× g for 90 min, the supernanant was discarded, the pellet was washed and resuspended in PBS. The optimal ratio of DBCO-DEXO and cRGD was explored using laser confocal colocalization. Fluorescent dye PKH67 is a novel dye that can fluorescently label exosomes. It can label exosomes by binding to lipid molecules in exosome membrane. The method of labeling DEXO and cRGD-DEXO with PKH67 is as follows: DiluentC: Combine 4 µL of PKH67 with 0.5 mL DiluentC to prepare a DiluentC solution, then mix 50 µL of PBS-suspended DEXO and cRGD-DEXO with 0.5 mL DiluentC, then add the PKH67-DiluentC solution to the samples. The mixture was incubated at 2~8℃ for 15~30min. 1 mL PBS solution containing 0.5% BSA was added to terminate the labeling reaction. The PKH67-labeled DEXO and cRGD-DEXO were re-isolated using ultracentrifugation. HCT-116 cells were added into confocal dishes at a density of 2×10 4 cells/mL. When the cell confluency reached 50%, 5 μg of PKH67-labeled DEXO and cRGD-DEXO were added into the dishes, followed by incubation at 37℃ in a 5% CO 2 atmosphere for 12, 36 and 48 h, respectively. Then, 2 μg/mL nuclear dye DAPI was added into the dishes, incubated at room temperature (RT) for 10 min, washed three times with PBS. Fresh complete DMEM culture medium was subsequently added, and the uptake of PKH67-labeled DEXO and cRGD-DEXO by HCT-116 cells at 12, 36, and 48 h were recorded. The colocalization coefficients of PKH67-labeled DEXO, cRGD-DEXO with HCT-116 cells were determined. 3.7 Construction and optimization of cRGD-DEXO/ORI 3.7.1 Explore the optimal ratio of cRGD-DEXO and ORI At RT, cRGD-DEXO and ORI were mixed thoroughly and incubated on a shaker at 200 rpm for 90min. To determine the optimal ratio of cRGD-DEXO to ORI, encapsulation efficiency (EE) and drug loading (DL) rate of ORI were used as evaluation criteria. The mass ratio of cRGD-DEXO to ORI was set at 10:1, 5:1, 1:1, 1:5 and 1:10 (µg:µg), respectively, and a single factor investigation was carried out. Finally, purified cRGD-DEXO/ORI was obtained through ultracentrifugation purification, and the ORI content in cRGD-DEXO/ORI was detected by an enzyme-assisted assay. EE (%)=(Wt-Wf)×100% Wt: the overall drug quality Wf: free drug quality DL (%)=We/Wm×100% We: carrier within the dosage Wm: the amount of drug carrier 3.7.2 Identification of cRGD-DEXO/ORI 20μL cRGD-DEXO/ORI suspension were added to a 2 mm copper grid, and the liquid was absorbed by filter paper. After standing at RT for 5 min, 10 μL of 3% amyl phosphate solution was added, followed by negative staining for 5 min. The grid was then baked under an incandescent lamp at 65℃ for 15 min before being photographed and observed. Before the test, 20μL of cRGD-DEXO/ORI suspension was diluted in 1 mL of ultrapure water and gently vortexed for 10 s to ensure homogeneous mixing. The diluted suspension was then shaken at 4℃ for 30 min. Before measurement, the particle size analysis was performed in triplicate using a particle size analyzer at RT, with each replicate measured 3 times to ensure data reliability. Key parameters including mean particle size and polydispersity index were recorded, and corresponding size distribution graphs were plotted. 3.8 Construction of cRGD-DEXO/ORI Based on the results obtained from the aforementioned experiment, cRGD-DEXO and ORI were mixed at a ratio of 1:1. ORI was loaded into cRGD-DEXO using an electroporation instrument at 300 v and 250 µF. The mixture was then ultracentrifuged at 11,8000× g for 90 min, the supernatant was discarded and the pellet was washed with PBS and then resuspended to obtain cRGD-DEXO/ORI. 3.9 cRGD-EXO/ORI safety range evaluation Normal liver cells LO2 were cultured of using the same protocol as HCT-116 cells. To evaluate the effects of cRGD-DEXO/ORI on LO2 cell viability, its concentration were set at 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 µg/mL, with 1 negative control groups included, resulting in 8 groups total. The cRGD-DEXO/ORI concentration at which the LO2 cell survival rate was ≥90% was selected for subsequent experiments. 3.1 0 Effects of cRGD-EXO/ORI on cells in vitro 3.10.1 Cell proliferation experiment The cellular state and confluency of HCT-116 cells were observed under an inverted fluorescence microscope. When the cell reached 80% confluency, cell passage and seeding were performed. HCT-116 cells were seeded into 96-well plates at a density of 1×10 5 cells /mL, with 200µL cell suspension added to each well. When the cell confluency reached approximately 70%, drug intervention was performed. The old medium was discarded, 200 µL PBS solution, 6.25 µg/mL ORI, 21.6 µg/mL DEXO, 21.6 µg/mL DEXO/ORI, 21.6 µg/mL cRGD-DEXO/ORI and 3 µg/mL 5-FU solution were added to each well, respectively. The plates were then incubated in CO 2 incubator at 37℃ for 24 h. After incubation, 20 µL of MTT solution was added to each well, followed by incubation in the dark for 4 h. The old medium was discarded and 150 µL of DMSO solution was added to each well, with gentle shaking for 10 min. The absorbance was measured at the wavelength of 490 nm, and the data were recorded. 3.10.2 Cell apoptosis was detected by acridine orange-ethidine bromide staining Acridine orange dye solution (reagent A), ethidine bromide dye solution (reagent B) and dilution buffer solution (reagent C) were mixed and diluted at a ratio of A:B:C=1:1:8 to prepare the working AO/EB staining solution. HCT-116 cells were digested using conventional method, then centrifuged at 1000 rpm for 5 min. The cell precipitate was collected, washed twice with PBS, and resuspended in dilution buffer (reagent C). The cell density was adjusted to (0.5~5)×10 6 cells /mL, and 25~50 µL cell suspension was used for subsequent staining. 2 µL of AO/EB staining solution was added, and the mixture was incubated at RT for 5~15min in the dark. After incubation, 5~10µL of stained cell suspension was dropped onto a glass slide, followed by observation and counting under a fluorescence microscope. 3.10.3 cell scratch test The horizontal lines were evenly drawn on the back of the 6-well plates with a ruler-guided marker pen (≥5 lines/well). HCT-116 cells were starved in serum-free medium for 12 h to eliminate serum interference. At 80% confluency, cells were washed 3 times with PBS, digested with trypsin, centrifugation post-termination with complete medium; supernatant removed, and the pellet was washed twice with PBS. The cells were resuspended in serum-free medium and seeded into pre-marked 6-well plates at 1×10 6 cells/well. After full adherence, the horizontal scratches (perpendicular to pre-drawn lines) were made with a 200 µL pipette tip; wells cleaned 3 times with PBS to remove detached cells. PBS solution, DEXO, ORI, DEXO/ORI, cRGD-DEXO/ORI and 5-FU solution were added to respective wells. Images were taken at 0 and 24 h post-treatment (3 random fields/well). Three fields of view were found, and image J software was used for analysis to calculate the mean value of relative area of intercellular migration. 3.11 Effects of cRGD-EXO/ORI on subcutaneous tumors in nude mice 3.11.1 Preparation and storage of drugs 2g of carboxymethyl cellulose sodium (CMC-Na) was weighed and added to 398 mL of deionized double steam water. Although CMC-Na can be dissolved in both cold and hot water, it is not easy to disperse evenly when mixed directly with cold water. Thus, the powder was first pre-wetted with a small volume of hot water and stirred thoroughly to form a homogeneous paste, after which the remaining hot water was added. And then, the mixture was subjected to thorough stirring and sonication to promote rapid expansion and dissolution of the paste. Finally, the solution was left overnight to ensure complete stabilization. 5-FU was dissolved in PBS under sterile conditions and diluted to a final concentration of 30 µg/mL. D-luciferin potassium salt, a common substrate for luciferase, is widely used in the whole field of biotechnology, especially in vivo imaging technology. The D-lucifcein potassium salt freeze-dried powder was first allowed to equilibrate to RT. It was then dissolved in Dulbecco's Phosphate-Buffered Saline(DPBS) to a final concentration of 15 mg/mL, followed by filtration with a 0.22 µm filter for sterilization. The resulting solution was divided into packages and stored at -20℃ in the dark. 3.11.2 Establishment of transplant tumor model in BALB/c nude mice HCT-116-LUC cells are bioluminescent CRC cells that bind to the substrate D-fluoresce in potassium salt in vivo , so that tumor growth can be observed in real time via small animal imaging. These cells were cultured using the same protocol as parental HCT-116 cells. Briefly, HCT-116-LUC cells in the logarithmic growth stage were collected, centrifuged, washed twice with serum-free DMEM medium, and resuspended for subsequent cell counting. The cell suspension was diluted with DMEM to a density of 2×10 7 cells/mL. BALB/c nude mice were provided by Beijing Huafukang Biotechnology Co., LTD. [SCXK (Beijing) 2019-0008] and housed in the Animal Experimental Research Center of Henan University of Chinese Medicine [SYXK (Henan) 2021-0015]. All animal procedures were approved by the Experimental Animal Ethics Committee of Henan University of Chinese Medicine (Approval No.: IACUC-202308018). Prior to injection, the skin at injection site (right axillary) of nude mice was disinfected with complex iodine. A 0.1 mL of cell suspension (containing about 2×10 6 cells) was injected subcutaneously into the right axillary area. To ensure uniform cell distribution, the cell suspension was thoroughly mixed before extraction. After the injection, the injection site was gently pressed with a sterile cotton swab for 1 min to prevent leakage. The growth of transplanted tumor was monitored every other day from day 2 post-tumor cell implantation. The long diameter (a) and short diameter (b) of the transplanted tumor were measured with vernier calipers, and the volume of transplanted tumor V= (a•b2)/2 was calculated. On day 7 post-implantation, the subcutaneous nodules with a diameter of 3~6 mm diameter and hard texture were visible to the naked eye at the injection site, which were identified as tumors. Tumor volumes were measured, tumor formation rate was calculated, each mouse was weighed with a balance, weight distribution were recorded and analyzed. Mice with disabilities, poor health status, or weight/tumor volume outside the range were excluded. The remaining nude mice were collectively housed in a cage, allowed to move freely for 10 min, then randomly assigned to experimental groups and numbered for documentation. 3.11.3 Drug administration of BALB/c nude mice The nude mice were divided into model group, ORI group, cRGD-DEXO/ORI group and 5-FU group, with 6 mice in each group. Following grouping, the volume of transplanted tumors in each nude mouse was measured again, and inter-group differences were subjected to statistically analyzed. The first day of administration was recorded as day 0. Each group was injected with 0.9% normal saline, 5 mg/kg ORI, 8.6 mg/kg cRGD-DEXO/ORI and 25 mg/kg 5-FU via tail vein every three days for a total of 6 administrations (18 days). Before each administration, the body weight of nude mice was measured using an electronic balance, and the long diameter (a) and short diameter (b) of subcutaneous tumors were measured with a vernier caliper. The graft volume V=(a·b2)/2 was calculated. To evaluate the effects of different drug treatments on the growth of transplanted colorectal cancer tumors, line graphs were plotted with time as the x-axis and tumor volume and body weight of nude mice as the y-axis respectively. 3.11.4 Fluorescence detection analysis Luciferase-encoding sequences are delivered to target cells or tissues via a delivery to enable luciferase expression. When corresponding substrate is introduced through intraperitoneal or intravenous injection, enzyme catalyzes luciferin oxidation to produce luminescence within a few minutes under the participation of ATP and oxygen. Notably, luminescence phenomenon occurs exclusively in living tissues, and there is a linear relationship between the intensity of luminescence and number of labeled cells. For experimental measurements, nude mice received intraperitoneal injections of 200 µL D-luciferin potassium salt on day 0 and every 3 days after post-administration, with injections administered 20 min before imaging, mice then were anesthetized with isoflurane gas and photographed using bioluminescence mode of live imaging of small animals. To analyze the data, tumor bioluminescence intensity was plotted against time (x-axis: time; y-axis: tumor bioluminescence intensity). Statistical analysis was performed on group data, with outliers (data points outside the range of mean±2 standard deviations) excluded. Tumor bioluminescence kinetic curves were then generated using the mean values of the remaining data. 3.11.5 HE staining analysis At the end of dosing cycle, orbital blood extraction was used to rapidly drop blood into the 1.5 mL EP tube. The right axillary skin of nude mice was cut on the ice plate, the tumor tissue was carefully removed, weighed and photographed, and then divided into three equal parts for HE and WB detection, respectively. The heart, liver, spleen, lung, kidney and other organs were simultaneously removed for subsequent HE detection. Tumor inhibition rate of each group=(average tumor volume of control group-average tumor volume of experimental group)/average tumor volume of control group×100%. Then tumor tissues and nude mouse organs were fixed in 10% neutral-buffered formalin for 24 h. After dehydration through graded ethanol, clearing in xylene, and infiltration with paraffin wax, samples were embedded in paraffin blocks. Sections (4~6 μm) were cut, deparaffinized, and rehydrated. Subsequently, they were stained with hematoxylin for 5~10 min, differentiated in acid alcohol, blued, and then stained with eosin for 1~2 min. After a final dehydration, clearing, and mounting, the slides were examined under a light microscope. 3.12 Statistical analysis The experimental data were collected, and normality and homogeneity of variance were tested by SPSS 25.0 statistical software. If the data conform to the normal distribution and the variance is homogeneous, one-way ANOVA is used, and LSD statistical method is used for pound-by-pair comparison. The variance was not uniform, and Dunnett's T3 statistical method was used for comparison between groups. If the data does not conform to the normal distribution, the non-parametric test is used. GraphPad Prism 9 processes bar graphs, in which P <0.05 indicated that the difference was statistically significant. 4. Discussion In recent years, many plant compounds have been proved to be good for disease and health, and more and more new active ingredients of Chinese medicine are effective against tumors. However, due to the poor targeting of some natural compounds, their clinical application is greatly limited. With the rise of new nanotechnology, combining these natural compounds with nanotechnology-based drug delivery systems to prepare TCM compound nanopreparations may further improve the clinical effectiveness of these drugs. Accordingly, in this study, we selected ORI a natural compound with potent anti-CRC activity and loaded it into DEXO. To improve targeting specificity, we functionalized DEXO with cRGD peptides, constructing a targeted drug delivery system (cRGD-DEXO/ORI) for CRC. Our findings confirmed that this system exhibits robust targeting to CRC cells and exerts significant anti-CRC effects both in vitro and in vivo . Notably, this study addresses two critical challenges in natural compound-based cancer therapy: poor targeting and limited bioavailability. By leveraging DEXO as a novel carrier, we not only harness the biocompatibility and natural cargo-loading capacity of exosomes but also exploit the unique advantages of Ds (e.g., low immunogenicity, scalable production) to overcome the limitations of conventional nanocarriers. The conjugation of cRGD further enhances tumor-specific delivery by exploiting the high expression of integrin αvβ3 in CRC, thereby minimizing off-target effects and improving therapeutic precision. Collectively, these results validate cRGD-DEXO/ORI as a viable platform to augment the anti-CRC efficacy of ORI, providing a rationale for its potential clinical application. Because ORI has water solubility, poor absorption rate and difficulty in penetrating the blood-brain barrier, we adopted DEXO to wrap ORI to overcome these shortcomings. DBCO, as an amine reactive NHS ester, has the ability to quickly and easily attach the reactive part to virtually any primary or secondary amine group, showing its potential for high efficiency and wide range of applications. In this study, DBCO was used as an intermediate to connect cRGD and DEXO to form cRGD-DEXO under certain reaction conditions. To verify the targeting of cRGD-DEXO to HCT-116 cells, PKH67 was used to label DEXO membrane. HCT-116 cells were cultured in vitro, and DEXO and cRGD-DEXO membranes were stained with PKH67 dye. After staining, they were added to culture dishes for 24 h. The results of confocal laser microscopy showed that compared with DEXO, cRGD-DEXO showed good targeting on CRC HCT-116 cells in vitro. Interestingly, with the extension of time, cRGD-DEXO gradually transferred from cell membrane to cytoplasm and nucleus, which may be due to the fact that cRGD is a tumor penetrating peptide. It can play a "perforating" role, and then penetrate into the tumor tissue [16] . Exosomes of different cellular origin preferentially interact with specific types of recipient cells, depending on the different classes of proteins they contain on their membranes. When exosomes bind to the surface of the recipient cell membrane, they act in two main ways. One is that proteins on the exosome membrane bind to and activate receptors on the recipient cell membrane, and the recipient cell does not take up exosomes [17] . The other is that exosomes deliver their contents into the recipient cell through direct fusion with the recipient cell membrane or endocytosis [18] . In this experiment, we played a role by targeting the delivery of cRGD-DEXO/ORI to HCT-116 cells, which bound to and internalized ORI. Studies have shown that this process is a receptor-mediated and energy-dependent active process, mainly dependent on the fossa and energy-dependent endocytosis pathways regulated by clathrin, which can be inhibited by mycotoxin and chlorpromazine [19] . The ideal drug delivery method should not only load drugs into exosomes, but also maintain the structural and functional integrity of exosomes and drugs [20] . At present, the common drug delivery strategies include exosomal pre-delivery and post-delivery. Pre-delivery is an effective drug delivery technique, but the drug delivery efficiency of this method is low. Another drug delivery strategy is post-secretion drug delivery, which has the advantage of high drug delivery efficiency and the disadvantage of damaging the integrity of exosome membrane [21] . After comprehensive evaluation, we choose electroporation method for ORI loading. In order to explore the loading rate and encapsulation rate of ORI and cRGD-DEXO at different ratios, ORI was loaded at 300v and 250µF according to the literature [22] . The results showed that with the increase of ORI concentration, the encapsulation rate showed a downward trend. The reason may be that when ORI concentration was too high, the fluidity of exosome membrane would be affected, and thus the encapsulation rate would decline, or the concentration of ORI reached the upper limit of the capacity that the exosome could sustain. In addition, when the "truck" concentration of exosomes increased, the drug loading increased sharply, but the encapsulation rate decreased slightly, resulting in a decrease in the utilization of exosomes. After comprehensive consideration, the ratio of ORI to cRGD-DEXO was 1:1 for subsequent experiments, and the loading rate and encapsulation rate were 22% and 29%, respectively. In addition, we also discussed the 1:1 ratio of ORI and cRGD-DEXO, different voltage capacitance conditions (voltage: 50, 100, 250, 300, 400 and 500v, capacitance: 125, 250 and 500µF) on ORI loading rate and packet rate, the results confirm that ORI loading rate and packet rate reach the best when the ratio of ORI to cRGD-DEXO is 1:1 at 300v and 250µF. In addition, after coupling cRGD and loading ORI, the DEXO particle size increases and is well distributed, and the potential decreases, which may be related to the negative charge of cRGD itself. In vitro experiments in this study confirmed that cRGD-DEXO/ORI had a good inhibitory effect on HCT-116 cells, and the results of cell proliferation and AO-EB staining showed that compared with the ORI group, the cell inhibition rate and apoptosis rate in the DEXO/ORI group were significantly increased. In addition, compared with DEXO/ORI group, The inhibition rate and apoptosis rate of cRGD-DEXO/ORI group were also significantly increased. The mechanism is as follows: On the one hand, ORI coated with DEXO greatly improves ORI's blood circulation time and ability to cross the blood-brain barrier. On the other hand, cRGD-DEXO/ORI can be well targeted to HCT-116 cells, which may be attributed to the high affinity of cRGD for integrin αvβ3. cRGD significantly increased the endocytosis efficiency of HCT-116 cells and increased the local effective concentration of HCT-116 cells. In vivo results showed that cRGD-DEXO/ORI also showed a good inhibitory effect on tumor growth, and had no effect on body weight in nude mice. In order to track the distribution and metabolism of exosomes in vivo, we will conduct dynamic tracing of exosomes in the following experiments. 5. Summary and prospect In this study, we successfully constructed a cRGD-DEXO/ORI drug delivery system targeting the colorectal site, and results demonstrated that cRGD-DEXO/ORI system has a better targeting ability to CRC cells both in vitro and in vivo ,which could be used as a potential effective therapeutic agent for cancer in future. However, there are still some shortcomings in this study. For example, we will need to verify the targeting of cRGD-DEXO/ORI in vivo . On the one hand, the production of exosomes is small, the extraction time is long, the extraction steps are complicated, and the scale and engineering extraction process are lacking, which greatly limits the application of exosomes. On the other hand, the drug loading of exosomes is low, and the drug loading method needs to be further explored. At the same time, exosomes of different cell origin carry different membrane proteins on their membrane surface, which gives them different biological characteristics. How to effectively select and purify exosomes is both an opportunity and a challenge for us. The commercialization of exosomes still has a long way to go, which requires more clinically relevant systematic evaluation and comprehensive systematic comparison with protocols such as liposomes. Declarations Author contributions All authors took part in writing, reviewing, and editing the manuscript. All authors reviewed the manuscript and approved it for publication. Baiyan Wang and Shuying Feng main contributed to research conception and design; Lei Wang, Chunguang Zhou and Zilong Wang performed all experiments. Aifang Li, Shuxuan Li and Yalan Li directed the project, performed statistical analysis, and interpreted data.Yujing Huangfu and Wei Chen prepared the figures, Shuying Feng contributed to the manuscript revision, and read and approved the final submitted version. The corresponding author Shuying Feng takes primary responsibility for communication with the journal and editorial office during the submission process, throughout peer review, and during publication. Eth ical approval Funding This work was funded by the National Natural Science Foundation of China (82402600), the Joint Funds of Science and Technology Research and Development Project of Henan Province (No. 232301420070), the basic research project of key scientific research projects of universities in Henan Province (No. 23ZX005), the Natural Science Foundation of Henan Province (232300421164), and the Key Research and Development Special Project of Henan Province (241111311200). Availability of data and materials Not applicable. Conflicts of Interest The authors declare no conflict of interest. References Yang W, Zheng H, Lv W, et al. Current status and prospect of immunotherapy for colorectal cancer. Int J Colorectal Dis. 2023; 38(1): 266. Tyagi G, Kapoor N, Chandra G, et al. 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The ins-and-outs of exosome biogenesis, secretion, and internalization. Trends Cell Biol. 2024; 34(2): 90-108. Huang C, Zhou Y, Feng X, et al. Delivery of engineered primary tumor-derived exosomes effectively suppressed the colorectal cancer chemoresistance and liver metastasis. ACS Nano. 2023; 17(11): 10313-10326. Tenchov R, Sasso JM, Wang X, et al. Exosomes─nature's lipid nanoparticles, a rising star in drug delivery and diagnostics. ACS Nano. 2022; 16(11): 17802-17846. Zeng H, Guo S, Ren X, et al. Current strategies for exosome cargo loading and targeting delivery. Cells. 2023; 12(10): 1416. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 Mar, 2026 Reviews received at journal 06 Mar, 2026 Reviews received at journal 17 Feb, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers invited by journal 08 Jan, 2026 Editor assigned by journal 24 Dec, 2025 Submission checks completed at journal 24 Dec, 2025 First submitted to journal 21 Dec, 2025 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. We do this by developing innovative software and high quality services for the global research community. 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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-8418021","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":573434136,"identity":"54162b29-2e6f-476c-8160-dca5131e9c94","order_by":0,"name":"Baiyan Wang","email":"","orcid":"","institution":"Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Baiyan","middleName":"","lastName":"Wang","suffix":""},{"id":573434140,"identity":"c7dc5b17-713f-4c26-bf16-6cd95060064e","order_by":1,"name":"Aifang Li","email":"","orcid":"","institution":"Henan University of Traditional Chinese 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08:00:11","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110651,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8418021/v1/8b8a27c15aefe0a278c79a07.html"},{"id":100116660,"identity":"6590ff7d-d460-4c53-857e-dd81b58302e2","added_by":"auto","created_at":"2026-01-13 08:00:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":187027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different concentrations ORI, BBR, BAI on the proliferation of HCT-116 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA1, A2\u003c/strong\u003e: Effect of different concentrations ORI on the proliferation of HCT-116 cells (24h, 48 h). \u003cstrong\u003eA3, A4\u003c/strong\u003e: Effect of different concentrations BBR on the proliferation of HCT-116 cells (24 h, 48 h). \u003cstrong\u003eA5, A6\u003c/strong\u003e: Effect of different concentrations BAI on the proliferation of HCT-116 cells (24h, 48 h). Compared with the negative control group, a: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05，b:\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01，c:\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001，d:\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001; Comparison between groups, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. \u003cstrong\u003eA7\u003c/strong\u003e: Effect of different concentrations ORI on morphology of HCT-116 cells (12 h)(100×); \u003cstrong\u003eA8\u003c/strong\u003e: Effect of 6.25µg/mL ORI on morphology of LO2 cells (top, 100×; bottom, 200×).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8418021/v1/0a92cc8da0b91ee90af36985.png"},{"id":100116659,"identity":"7910c9f8-233a-459a-81a4-1d0dd08cea99","added_by":"auto","created_at":"2026-01-13 08:00:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":611542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization and identification of DEXO and cRGD-DEXO/ORI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB1, B2\u003c/strong\u003e: Electron microscopic image of DEXO and cRGD-DEXO/ORI (scale:100nm). \u003cstrong\u003eB3, B4\u003c/strong\u003e: Potential distribution map of DEXO and cRGD-DEXO/ORI. \u003cstrong\u003eB5,\u003c/strong\u003e \u003cstrong\u003eB6\u003c/strong\u003e: Particle size distribution map of DEXO and cRGD-DEXO/ORI.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8418021/v1/46981332d6e59aacb1e26268.png"},{"id":100116667,"identity":"25638156-8c7f-4c37-b737-64b4c2eb1457","added_by":"auto","created_at":"2026-01-13 08:00:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":561447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction process of cRGD-DEXO/ORI.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8418021/v1/9e68351abedaa401397d821d.png"},{"id":100116662,"identity":"a4897177-bf23-4dac-88b7-42095d984efb","added_by":"auto","created_at":"2026-01-13 08:00:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1173224,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction and optimization of cRGD-DEXO/ORI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC1:\u003c/strong\u003e The binding rate of DEXO and cRGD in different proportions. \u003cstrong\u003eC2\u003c/strong\u003e: Loading and encapsulation efficiency of DEXO and ORI at different ratios. \u003cstrong\u003eC3\u003c/strong\u003e:Uptake of DEXO and cRGD-DEXO by HCT-116 cells (200×). \u003cstrong\u003eC4\u003c/strong\u003e: Uptake of cRGD-DEXO by HCT-116 cells at different time points (200×). \u003cstrong\u003eC5\u003c/strong\u003e: Effect of different concentrations of cRGD-DEXO/ORI on the proliferation of LO2 cells (Comparison between groups, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of different groups on proliferation, apoptosis, migration of HCT-116 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD1\u003c/strong\u003e: Effects of different groups on HCT-116 cells proliferation. \u003cstrong\u003eD2\u003c/strong\u003e: Effects of different groups treatment on morphological characteristics of apoptosis of HCT-116 cells (100×). \u003cstrong\u003eD3\u003c/strong\u003e: Histograms of apoptotic cell distribution after different groups treatment. \u003cstrong\u003eD4, D5\u003c/strong\u003e: Effects of different groups on HCT-116 cells migration (100×, scale=200µm). Compared with the negative control group, a: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05，b:\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01，c:\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001，d:\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001; Comparison between groups, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8418021/v1/6194d9958e0eeaa4ce795586.png"},{"id":100365574,"identity":"713e56a3-371b-443d-a433-65242ef8723c","added_by":"auto","created_at":"2026-01-16 07:55:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":535252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntitumor effect of different groups of drugs on ectopic CRC in nude mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE1\u003c/strong\u003e: Effect of different groups on body weight of nude mice. \u003cstrong\u003eE2\u003c/strong\u003e: Inhibition effect of different groups on transplanted CRC tumors in nude mice. \u003cstrong\u003eE3\u003c/strong\u003e: Effect of different groups on the growth of transplanted CRC tumors in nude mice (tumor fluorescence intensity). \u003cstrong\u003eE4\u003c/strong\u003e: Bioluminescence imaging of tumor growth in nude mice. \u003cstrong\u003eE5\u003c/strong\u003e: Tumor inhibition rate of different groups of drugs on nude mice. \u003cstrong\u003eE6\u003c/strong\u003e: Effect of different groups of drugs on tumor volume in nude mice (photos after sampling). Comparison between groups, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8418021/v1/6304cc3173c19f46409910a8.png"},{"id":100382287,"identity":"abddeb22-ea7c-45e4-892e-dae63d61bdd6","added_by":"auto","created_at":"2026-01-16 10:41:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4225007,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8418021/v1/0d3840e4-6200-4103-a110-31b60a473fc9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preparation of microalgae-derived exosomes drug delivery system loaded with oridonin and evaluation of anti-colorectal cancer effect","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEpidemiological data show an increasing of colorectal cancer (CRC) year by year. Although significant advancements in chemotherapy, radiotherapy and targeted therapy, the prognosis of advanced CRC remains poor, with a persistently low 5-year survival rate\u003csup\u003e[1]\u003c/sup\u003e. Traditional Chinese medicine (TCM) exhibits multi-target and multi-pathway properties in diseases treatment, which is an important means of cancer management in China, especially for advanced malignancies. Numerous plant-based compounds have been shown to have benefits for disease and health. With the rapid development of drug mechanism separation technology and evaluation methods, an increasing number of active ingredients of TCM have been identified and verified for anti-tumor efficacy\u003csup\u003e[2]\u003c/sup\u003e. For instance, studies have shown that oridonin (ORI)\u003csup\u003e[3]\u003c/sup\u003e, berberine (BBR)\u003csup\u003e[4]\u003c/sup\u003e and baicalin (BAI)\u003csup\u003e[5]\u003c/sup\u003e all had proliferation-inhibiting effects on CRC cell. However, their clinical application is always hindered by poor stability, inadequate targeting, short half-life and low bioavailability etc.\u003c/p\u003e\n\u003cp\u003eThe rapid development of nanotechnology has successfully enabled nanoparticles to be widely applied as drug delivery systems cancer therapy. The nanization of TCM components has shown great advantages in recent years. Exosome (EXO), natural extracellular vesicle with a diameter of 30~150 nm secreted by various cells, are composed of lipids, proteins, and RNAs\u003csup\u003e[6]\u003c/sup\u003e. As key mediators of intercellular communication, they play an important role in transmitting genetic and biochemical information between cells\u003csup\u003e[7]\u003c/sup\u003e, emerging as promising drug delivery carriers\u003csup\u003e[8]\u003c/sup\u003e. Their advantages include: (1) excellent biocompatibility, low immunogenicity and minimal long-term safety concerns\u003csup\u003e[9]\u003c/sup\u003e; (2) membrane proteins, lipids and polysaccharides conferring inherent homing properties and natural sites for artificial modification\u003csup\u003e[10]\u003c/sup\u003e; and (3) efficient penetration of biological barriers\u003csup\u003e[11]\u003c/sup\u003e. However, the high cost of EXO isolation from animal cells limits their scalability. Fortunately, marine algae produce diverse bioactive metabolites with reported anticancer properties, offering alternative sources for exosome production\u003csup\u003e[12]\u003c/sup\u003e. \u003cem\u003eDunaliella salina\u0026nbsp;\u003c/em\u003e(\u003cem\u003eDs\u003c/em\u003e)\u003cem\u003e,\u003c/em\u003e a unicellular eukaryotic green algae thriving in bracken water, possesses remarkable natural advantages for genetic engineering and dpractical applications. Firstly, it is non-toxic, nutrient-rich, and enhances immune function and resistance as a natural health product\u003csup\u003e[13]\u003c/sup\u003e. Secondly, its lack of a cell wall (natural protoplast state) facilitates foreign genes transfection, holding promise for genetic engineering\u003csup\u003e[14]\u003c/sup\u003e. Compared to exosomes from other sources, \u003cem\u003eDs\u0026nbsp;\u003c/em\u003eexosomes (DEXO) offer high modifiability, stability transformation, low endogenous toxicity, and scalable production potential. Furthermore, DEXO include intrinsic anticancer agents such as polysaccharides and β-carotene\u003csup\u003e[15]\u003c/sup\u003e, which can play a synergistic role as drug carriers.\u003c/p\u003e\n\u003cp\u003eIn this study, cRGD was conjugated to DEXO membranes via chemical click reaction, and ORI was loaded into cRGD-DEXO using electroporation to construct an targeted drug delivery system (cRGD-DEXO/ORI) for CRC. Its anti-CRC efficacy was evaluated both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. This research holds dual significance: (1) it pioneers the application of DEXO as drug delivery vectors, providing theoretical and methodological insights for further carrier development; (2) it enhances the anti-CRC efficacy of ORI, offering strategies for clinical application of ORI.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003e2.1 Effects of ORI, BBR and BAI on the activity of HCT-116 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 1 A1, MTT results showed that after ORI was treated on HCT-116 cells for 24 h, ORI exhibited dose-dependent antiproliferative effects across concentrations of 6.25, 12.5, 25, 50, 100, and 200 µg/mL relative to the negative control group. All tested concentrations exerted statistically significant inhibitory effects on cell proliferation(\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001). A clear dose-dependent decrease in cell survival rate was observed: specifically, survival rate declined significantly as ORI concentrations increased from 3.125 to 25 µg/mL, dropping to approximately 3% at 25 µg/mL and remaining unchanged with further increases in ORI concentration (up to 200 µg/mL). For 48h ORI treatment, HCT-116 cells displayed a similar dose-dependent trend in cell survival rate as the 24h group. At 12.5 µg/mL ORI, cell survival rate was only 9.19%, and no further significant decrease was noted with higher concentrations. Furthermore, the antiproliferative effects of ORI at concentrations of 12.5, 25, 50, 100, and 200 µg/mL after 48 h were not significantly different from those observed at 24 h.\u003c/p\u003e\n\u003cp\u003eTreatment of HCT-116 cells with BBR for 24 h showed no significant inhibitory effect at concentrations less than 50 µg/mL (vs. negative control). Only at concentrations greater than or equal to 50 µg/mL did BBR exert inhibition, with cell viability remaining 76% at 200 µg/mL (Figure 1A3). After 48 h of BBR treatment, strong proliferation inhibition was observed at concentrations greater than or equal 6.25 µg/mL, cell viability dropped to 37% at 200 µg/mL (Figure 1A4). For BAI: After 24 h, cell viability was unaffected at concentrations less than 50 µg/mL (vs. negative control). Viability began to decline at 50 µg/mL, remaining 72% at 200 µg/mL. After 48 h, BAI had no inhibitory effect at concentrations less than 50 µg/mL. However, unlike the 24 h group, cell viability decreased to 43% at 200 µg/mL (Figure 1A6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Effect of ORI on the morphology of HCT-116 and LO2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter HCT-116 cells were treated with ORI at concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125 µg/mL for 24 h, significant morphological changes were observed. With increasing ORI concentration, cells gradually transformed from a spindle-shaped to a rounded morphology. Notably, most cell exhibited membrane budding, resembling apoptotic bodies (Figure 1, A7). To verify whether the ORI concentration that inhibits HCT-116 cells proliferation by 70% (IC₇₀) induces cytotoxicity on normal liver LO2 cells, we exposed LO2 cells to this IC70 concentration (Figure 1, A8) and the results demonstrated that compared with the untreated LO2 cells control group, LO2 cells treated with 6.25 µg/mL showed a long spindle-shape morphology, maintained a healthy growth state with uniform distribution and high density.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Characterization and identification of DEXO and cRGD-DEXO/ORI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTEM revealed that DEXO exhibited a \"saucer-like\" structure with intact morphology, consistent with the established morphological features of typical exosome (Figure 2, B1). In contrast, cRGD-DEXO/ORI displayed a \"spherical\" structure and maintained structural completeness (Figure 2, B2). Zeta potentials and particle size distributions of DEXO and cRGD-DEXO/ORI were measured by a DLS analyzer. Results showed that both DEXO (Figure 2, B3) and cRGD-DEXO/ORI (Figure 2, B4) exhibited zeta potentials ranging from -30~-50 mv, with cRGD-DEXO/ORI showing a modest reduction in surface charge compared to DEXO. Regarding particle size, DEXO was predominantly distributed within the range of 90~140 nm (Figure 2, B5), whereas cRGD-DEXO/ORI displayed a slight increase in size, with a broader distribution spanning 92~295 nm and a main peak concentrated at 164~255 nm (Figure 2, B6). Both samples exhibited PDI. Collectively, All these findings confirm the successful isolation of DEXO and demonstrate that both DEXO and cRGD-EXO/ORI conform to the established physicochemical characteristics of exosomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Construction and optimization of cRGD-DEXO/ORI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 3 C1, blue fluorescence (DAPI staining) indicates the nuclei of HCT-116 cells, while green fluorescence (PKH67 labeling) corresponds to DEXO. The merged image demonstrates colocalization of DEXO and HCT-116 cells. CLSM revealed that PKH67-labeled DEXO and cRGD-DEXO localized to both the cell membrane and cytoplasm of HCT-116 cells. Notably, compared with unmodified DEXO, cRGD-DEXO exhibited significantly stronger fluorescence intensity in their cell membrane and cytoplasm. These results indicate that cRGD modification enhances the targeting ability of DEXO to HCT-116 cells and promotes their internalization.\u003c/p\u003e\n\u003cp\u003eFigure 3 C2 illustrates the time-dependent uptake of cRGD-DEXO by HCT-116 cells at different time points. CLSM showed that at 12h, numerous green fluorescent dots (PKH67-labeled cRGD-DEXO) formed ring-like distributions around the cell membrane and nucleus. With extended incubation, green fluorescence signals also appeared within the nucleus. This observation was attributed to the tumor-penetrating properties of cRGD, potentially via a \"hole\" effect. Collectively, these results indicated that cRGD-DEXO not only targets the cell membrane of HCT-116 cells but also undergoes internalization, with intracellular accumulation increasing over time. In addition, as shown in Figure 3 C3, the binding ability of DEXO was optimal at a DEXO to cRGD ratio of 5:1. Therefore, the cellular uptake of cRGD-DEXO by HCT-116 cells exhibited a time-dependent increase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Establishment of ORI standard curve\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 3 C4, analysis the relationship between ORI concentration and absorbance at 238 nm yielded a standard curve equation of y=0.003x+3.34 (R2=0.9837), spanning a linear range of 0.78125~25 µg/mL. These results demonstrate a strong linear correlation between ORI concentration and absorbance at 238 nm, which is sufficient to meet the requirements for quantitative analysis and subsequent experiments procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Determination of loading rate and encapsulation rate of cRGD-DEXO and ORI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 3 C5, the loading rate and encapsulation rate of ORI were quantified using a microplate reader at different ratios of cRGD-DEXO and ORI. The results demonstrated that increasing ORI concentration led to a downward trend in encapsulation rate, while the loading rate stabilized. Conversely, increasing cRGD-DEXO concentration resulted in an upward trend in the encapsulation rate accompanied by a slight decrease in loading rate. At an ORI:cRGD-DEXO ratio of 1:1, the loading rate was 22.3% and encapsulation rate was 29%, representing an optimal balanced that maximized effective utilization of both components. Therefore, the 1:1 ORI: cRGD-DEXO of ratio was selected for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 cRGD-DEXO/ORI safety range evaluation and its effect on HCT-116 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the biocompatibility of cRGD-DEXO/ORI, LO2 cell line was used as an \u003cem\u003ein vitro\u003c/em\u003e model to assess its safe concentration range. LO2 cells were treated with different concentrations cRGD-DEXO/ORI, and the effect of cRGD-DEXO /ORI on LO2 cells viability was detected by MTT assay. As shown in Figure 3 C6, after 24 h co-incubation of cRGD-DEXO/ORI with the cells, no inhibitory effect on LO2 cells proliferation was observed with cell viability remaining above 80%. However, when the concentration of cRGD-DEXO/ORI reached 500 µg/mL, cell viability dropped below 80%.To evaluate the antiproliferative efficacy of cRGD-DEXO/ORI \u003cem\u003ein vitro\u003c/em\u003e, HCT-116 cells were treated with different formulations for 24 h, and cell viability was analyzed. As shown in Figure 4 D1, the cell survival rate in DEXO/ORI group was significantly lower than that in ORI group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Furthermore, the cell proliferation inhibition ability of cRGD-DEXO/ORI group was stronger than that of DEXO/ORI group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). However, no significant difference was observed between the cRGD-DEXO/ORI and 5-FU groups. In addition, DEXO group showed no inhibitory effect on proliferation of HCT-116 cells, with cell survival rate remaining as high as 97%. Notably, ORI encapsulated in DEXO (i.e., DEXO/ORI) exerted a potent inhibitory effect on cell proliferation: compared with ORI group, cell survival rate in DEXO/ORI group was reduced by approximately 1.6-fold. Following modification of DEXO/ORI with cRGD, cell survival rate was reduced by 1.85-fold compared with DEXO/ORI group. Overall, cRGD-DEXO/ORI intervention resulted in a significant 5-fold reduction in HCT-116 cell viability relative to the negative control group. In conclusion, cRGD-DEXO/ORI exhibited favorable\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e inhibitory activity against CRC cells proliferation, and ORI encapsulated in cRGD-modified DEXO showed enhanced antiproliferative efficacy compared to ORI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Effects of cRGD-DEXO/ORI on apoptosis of HCT-116 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAO can penetrate complete cell membranes and intercalate into nuclear DNA, exhibiting bright green fluorescence. In contrast, EB can only permeates cells with compromised membranes and intercalates into nuclear DNA, emitting orange fluorescence. Under the fluorescence microscope, four different cell morphology can be clearly distinguished: viable cells exhibit green and maintains a normal structure. The chromatin of early apoptotic cells also showed green fluorescence but with condensed or beaded fragmented chromatin. In contrast, non-apoptotic dead cells show orange-stained chromatin that maintains normal structure, whereas the chromatin of late apoptotic cells present orange-red chromatin with pronounced condensation or fragmentation. These morphological differences provide an intuitive and accurate method for assessing cellular status. As shown in Figure 4 D2, D3 and D4, the late apoptosis rate of HCT-116 cells in the cRGD-DEXO/ORI group was statistically significantly increased compared with the negative control group. The late apoptosis rates accounted for 21%, 63%, 75% and 64% of total cells in ORI, DEXO/ORI, cRGD-DEXO/ORI and 5-FU groups, respectively. In contrast, cell in the negative control group and DEXO groups exhibited normal growth, with dead cells comprising only 9% and 11% of total cell population, respectively. In conclusion, cRGD-DEXO/ORI markedly enhances the apoptosis rate of HCT-116 cells, primarily inducing late apoptotic phenotypes, whereas DEXO alone has no inhibitory effect on HCT-116 cells growth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Effects of cRGD-DEXO/ORI on HCT-116 cells migration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 4 D5 and D6 demonstrate the effects of different drugs groups on HCT-116 cell migration after 24 h. The results showed that the relative migration area in the DEXO/ORI, cRGD-DEXO/ORI and 5-FU groups was significantly lower than that in negative control group. Compared with ORI alone, the inhibition effect of cRGD-DEXO coated ORI on HCT-116 cells migration was approximately twice as potent. Notably, the relative migration area in cRGD-DEXO/ORI group was the smallest, accounting for roughly 1/3 of that in negative control group. In conclusion, the DEXO/ORI, cRGD-DEXO/ORI and 5-FU groups all exerted inhibitory effects on HCT-116 cell migration, with the cRGD-DEXO/ORI group exhibiting the most pronounced inhibitory effect.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 \u003cem\u003eIn vivo\u0026nbsp;\u003c/em\u003eanti-tumor evaluation of subcutaneous tumors in nude mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of cRGD-DEXO/ORI on tumor growth inhibition and body weight were evaluated in BALB/c nude mice. As shown in Figure 5 E1, compared with the model group, the ORI, cRGD-DEXO/ORI and 5-FU groups had different degrees of inhibition on CRC growth. Notably, the growth inhibition effect of cRGD-DEXO/ORI on CRC was more obvious than that of ORI alone or 5-FU groups. On day 18, the tumor volumes in model, ORI and 5-FU groups were 2.59-, 1.91-, and 1.38-fold those in cRGD-DEXO/ORI group, respectively. In addition, as shown in Figure 5 E2, the different drug treatments exerted no significant effect on the body weight of nude mice. Collectively, these results indicate that ORI exhibits potent anti-CRC activity, and this activity is significantly enhanced when ORI is encapsulated in cRGD-DEXO. As shown in Figure 5 E3 and E4, the change curves of tumor fluorescence intensity changes over time in nude mice with CRC xenografts treated with different drugs. The results demonstrated that tumor fluorescence intensity was enhanced to varying degrees over time. Meanwhile, on day 18, ORI (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), cRGD-DEXO/ORI (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01), and 5-FU (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) groups had weaker fluorescence intensity than the model group, with reductions of 1.27-, 1.42-, and 1.30-fold, respectively. In addition, ORI group fluorescence was 0.89-fold (vs cRGD-DEXO/ORI, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) and 0.98-fold times (vs 5-FU, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) that of latter two groups, with no significant difference between the 5-FU and the cRGD-DEXO/ORI groups. In view of this, cRGD-DEXO/ORI exhibits a certain capacity to inhibit CRC growth. Meanwhile, as shown in Figure 5 E5 and E6, tumor volume and tumor inhibition rate analyses revealed that the anti-CRC activity of cRGD-DEXO/ORI was superior to that of ORI group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Compared with ORI group, 5-FU exerted a more pronounced inhibitory effect on CRC growth (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Additionally, no significant difference in mean tumor weight was observed between the cRGD-DEXO/ORI group and the 5-FU group (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). The tumor inhibition rates in the ORI, cRGD-DEXO/ORI and 5-FU groups were 26%, 61% and 47%, respectively.\u003c/p\u003e"},{"header":"3. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e3.1 Culture of HCT-116 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman\u0026nbsp;CRC\u0026nbsp;HCT-116 cells were supplied by Shanghai Fuheng Material Technology Co., Ltd. (Shanghai, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37℃ under a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e, with medium refreshed every 48-72 h to ensure optimal nutrient supply. Cells were passaged at 90% confluency: spent medium was aspirated, cells rinsed three times with pre-warmed PBS (pH 7.4), then 2 mL 0.25% trypsin-EDTA added and incubated at 37°C for 2~3 min until rounded. Digestion was terminated with 3 mL complete DMEM (10% FBS), and cells were pipetted to a single-cell suspension, transferred to a 15 mL tube, centrifuged at 1000 rpm for 5 min. Supernatant was aspirated, pellet resuspended in fresh medium, seeded 1:3 into new T-75 flasks, and returned to the incubator for expansion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Comparison of antitumor activity of ORI, BBR and BAI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eORI, BBR and BAI were purchased from Chengdu Master Biotechnology Co., Ltd., and were prepared at 7 concentration gradients (200, 100, 50, 25, 12.5, 6.25, 3.125 µg/mL, alongside a negative control group and a blank control group. The required volume of drug working solution was calculated, and solution were diluted to target concentrations using DMEM containing 10% FBS. 96-well plates were removed from the CO\u003csub\u003e2\u003c/sub\u003e incubator, original medium were discarded, and drug-containing media at gradient concentrations were added to wells (200 µL per well). Each concentration was tested in 6 replicate wells, and plates were labeled with date, time and dosage. Incubation was continued in the CO\u003csub\u003e2\u003c/sub\u003e incubator for 24 and 48h, respectively. After incubation, medium was aspirated, and 200 µL of pre-prepared 5 mg/mL MTT solution was added to each well (protected from light). Following 4 h of incubation, MTT solution was discarded, and 150 µL DMSO was added to dissolve blue-violet formazan crystals (produced by succinic dehydrogenase-mediated reduction of MTT in viable cells). Plates were shaken for 10 min to ensure complete crystal dissolution. Absorbance (OD) at 490 nm was measured using a microplate reader. Cell survival rate=(experimental group OD-blank group OD)/(control group OD-blank group OD)×100%. The experiment was repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Effects of ORI on the morphology of HCT-116 and LO2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHCT-116 cells culture and plate seeding were performed as described above. ORI was added to HCT-116 cells at concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125 µg/mL. After 24 h of treatment, cells were observed and photographed under an inverted microscope. Culture of normal hepatocyte LO2 (obtained from the First Clinical School of Medicine) and plate seeding were performed as those for HCT-116 cells. When cell density reached 70%, 200µL of 6.25 µg/mL ORI was added to each well. After 24 h, the morphology of LO2 cells was observed under an inverted fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Culture of \u003cem\u003eDs\u003c/em\u003e cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare the medium, 950 mL of distilled water was first measured, and the following reagents were sequentially dissolved in it: NaNO\u003csub\u003e3\u003c/sub\u003e (75 g/L), NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e· H\u003csub\u003e2\u003c/sub\u003eO (5 g/L), Na\u003csub\u003e2\u003c/sub\u003eSiO\u003csub\u003e3\u003c/sub\u003e·9H\u003csub\u003e2\u003c/sub\u003eO (30 g/L), CuSO\u003csub\u003e4\u003c/sub\u003e·5H\u003csub\u003e2\u003c/sub\u003eO (9.8 g/L), Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e·2H\u003csub\u003e2\u003c/sub\u003eO (6.3 g/L) and ZnSO\u003csub\u003e4\u003c/sub\u003e·7H₂O(22.0 g/L), CoCl\u003csub\u003e2\u003c/sub\u003e·6H\u003csub\u003e2\u003c/sub\u003eO (10.0 g/L), MnCl\u003csub\u003e2\u003c/sub\u003e·4H\u003csub\u003e2\u003c/sub\u003eO (180.0 g/L), Vitamin B12 (1 mg/mL), Biotin (0.1 mg/mL). The solution was then quantified to 1L with distilled water, and the pH was adjusted to 8 using NaOH before autoclaving for sterilization. \u003cem\u003eDs\u003c/em\u003e cells were retrieved from cryovials and quickly thawed in a 37°C water bath and then carefully and slowly added to the PKS liquid medium using a sterile pipette. The cells were cultured overnight in an illuminated incubator at 26°C with a 14:10 light/dark cycle. The following day, \u003cem\u003eDs\u0026nbsp;\u003c/em\u003ecell concentration was quantified using a visible-light spectrophotometer, and 50 μL cell suspension (1×10\u003csup\u003e6\u003c/sup\u003e cells /mL) was plated onto PKS solid medium by sterile pipette. Cultivation was continued under the same conditions (26°C, 14:10 light/dark cycle) until distinct monoclonal colonies had formed. Individual colonies were carefully selected and transferred to fresh PKS solid medium, then scaled up to 1 L PKS liquid medium for further cultivation. After incubation 3 d, when the cell concentration reached 1×10\u003csup\u003e6\u003c/sup\u003e cells/mL, cell expansion was performed. Finally, the 2nd to 4th generation of \u003cem\u003eDs\u003c/em\u003e cell cultures were selected as high-quality starting material for extracting exosome extraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Extraction and characterization of DEXO\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.5.1 Extraction of DEXO\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter 7 d of culture, the \u003cem\u003eDs\u0026nbsp;\u003c/em\u003ecell culture medium was collected for DEXO extraction by ultracentrifugation, following these steps: First, the medium was centrifuged at 4°C, 2,000×\u003cem\u003eg\u003c/em\u003e for 10 min to remove intact algal cells; the resulting supernatant was then centrifuged at 4°C, 10,000×\u003cem\u003eg\u003c/em\u003e for 30 min to remove cell debris. Next, the clarified supernatant was ultracentrifuged at 4°C, 11,8000×\u003cem\u003eg\u003c/em\u003e for 90 min. After discarding the supernatant, the exosomes-enriched pellet adhering to the ultracentrifuge tube wall was resuspended in PBS, filtered with 0.22 µm filter membrane, and ultracentrifuged again at 4°C, 11,8000×\u003cem\u003eg\u003c/em\u003e for 90 min. The final supernatant was resuspended in a small amount of PBS solution to obtain DEXO. Protein concentration of DEXO was quantified using the BCA method. If the concentration was too low, additional ultracentrifugation was performed for further enrichment. Aliquots were stored at -80°C, with a storage period of 6 months, and repeated free-thaw cycles were avoided.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.5.2 Characterization of DEXO\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor transmission electron microscopy (TEM) analysis, parafilm was cut into sections, and a copper mesh was placed onto the droplet with tweezers for 5 min incubation. Excess sample was blotted with filter paper, and 10 µL 3% uranyl acetate was dropped on a fresh parafilm section. The copper mesh was transferred onto the staining solution, excess stain was blotted, and the mesh was air-dried. Finally, the copper mesh was imaged via TEM with micrographs captured for documentation. Open the machine according to the instructions, and use a 1mL pipette to move the sample into the cuvette. The sample volume is equal to the scale on the cuvette. After that, cuvette was inserted into the instrument’s sample compartment and measurements were initiated. If the polydispersity index (PDI) exceeded the acceptable threshold, sample concentration may be too high or agglomeration. Appropriate dilution or ultrasonic treatment was performed as necessary, the measurement is repeated three times, with experimental data recorded and corresponding graphs generated for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Construction and optimization of cRGD-DEXO\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDBCO is a reactive cyclooctyne used for strain-promoted azides-alkyne cycloaddition, which can react with substances containing primary or secondary amino groups without copper. 1mg/mL DBCO solution was added to 1mg/mL DEXO solution, and the mixture was incubated on a mixer at 4℃ and pH 7.4 for 4 h. After incubation, the mixture was centrifuged at 16,4000×\u003cem\u003eg\u003c/em\u003e for 90 min, the supernatant was discarded, and the pellet then washed and resuspended in PBS to obtain DBCO-DEXO. The protein concentration was measured using a BCA assay. For optimizing the ratio of DBCO-DEXO to cRGD, mass ratios of 1:1, 1:2, 1:5, 2:1 and 5:1 (μg:μg) were set, and the reaction was incubated on a rotary mixer at 4℃ and pH 7.4 for 12 h. The mixture was then centrifuged at 16,4000×\u003cem\u003eg\u003c/em\u003e for 90 min, the supernanant was discarded, the pellet was washed and resuspended in PBS. The optimal ratio of DBCO-DEXO and cRGD was explored using laser confocal colocalization. Fluorescent dye PKH67 is a novel dye that can fluorescently label exosomes. It can label exosomes by binding to lipid molecules in exosome membrane. The method of labeling DEXO and cRGD-DEXO with PKH67 is as follows: DiluentC: Combine 4 µL of PKH67 with 0.5 mL DiluentC to prepare a DiluentC solution, then mix 50 µL of PBS-suspended DEXO and cRGD-DEXO with 0.5 mL DiluentC, then add the PKH67-DiluentC solution to the samples. The mixture was incubated at 2~8℃ for 15~30min. 1 mL PBS solution containing 0.5% BSA was added to terminate the labeling reaction. The PKH67-labeled DEXO and cRGD-DEXO were re-isolated using ultracentrifugation.\u003c/p\u003e\n\u003cp\u003eHCT-116 cells were added into confocal dishes at a density of 2×10\u003csup\u003e4\u003c/sup\u003ecells/mL. When the cell confluency reached 50%, 5 μg of PKH67-labeled DEXO and cRGD-DEXO were added into the dishes, followed by incubation at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere for 12, 36 and 48 h, respectively. Then, 2 μg/mL nuclear dye DAPI was added into the dishes, incubated at room temperature (RT) for 10 min, washed three times with PBS. Fresh complete DMEM culture medium was subsequently added, and the uptake of PKH67-labeled DEXO and cRGD-DEXO by HCT-116 cells at 12, 36, and 48 h were recorded. The colocalization coefficients of PKH67-labeled DEXO, cRGD-DEXO with HCT-116 cells were determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Construction and optimization of cRGD-DEXO/ORI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.7.1 Explore the optimal ratio of cRGD-DEXO and ORI\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAt RT, cRGD-DEXO and ORI were mixed thoroughly and incubated on a shaker at 200 rpm for 90min. To determine the optimal ratio of cRGD-DEXO to ORI, encapsulation efficiency (EE) and drug loading (DL) rate of ORI were used as evaluation criteria. The mass ratio of cRGD-DEXO to ORI was set at 10:1, 5:1, 1:1, 1:5 and 1:10 (µg:µg), respectively, and a single factor investigation was carried out. Finally, purified cRGD-DEXO/ORI was obtained through ultracentrifugation purification, and the ORI content in cRGD-DEXO/ORI was detected by an enzyme-assisted assay.\u003c/p\u003e\n\u003cp\u003eEE (%)=(Wt-Wf)×100% Wt: the overall drug quality Wf: free drug quality\u003c/p\u003e\n\u003cp\u003eDL (%)=We/Wm×100% We: carrier within the dosage Wm: the amount of drug carrier\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.7.2 Identification of cRGD-DEXO/ORI\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e20μL cRGD-DEXO/ORI suspension were added to a 2 mm copper grid, and the liquid was absorbed by filter paper. After standing at RT for 5 min, 10 μL of 3% amyl phosphate solution was added, followed by negative staining for 5 min. The grid was then baked under an incandescent lamp at 65℃ for 15 min before being photographed and observed. Before the test, 20μL of cRGD-DEXO/ORI suspension was diluted in 1 mL of ultrapure water and gently vortexed for 10 s to ensure homogeneous mixing. The diluted suspension was then shaken at 4℃ for 30 min. Before measurement, the particle size analysis was performed in triplicate using a particle size analyzer at RT, with each replicate measured 3 times to ensure data reliability. Key parameters including mean particle size and polydispersity index were recorded, and corresponding size distribution graphs were plotted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 Construction of cRGD-DEXO/ORI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the results obtained from the aforementioned experiment, cRGD-DEXO and ORI were mixed at a ratio of 1:1. ORI was loaded into cRGD-DEXO using an electroporation instrument at 300 v and 250 µF. The mixture was then ultracentrifuged at 11,8000×\u003cem\u003eg\u003c/em\u003e for 90 min, the supernatant was discarded and the pellet was washed with PBS and then resuspended to obtain cRGD-DEXO/ORI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.9 cRGD-EXO/ORI safety range evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNormal liver cells LO2 were cultured of using the same protocol as HCT-116 cells. To evaluate the effects of cRGD-DEXO/ORI on LO2 cell viability, its concentration were set at 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 µg/mL, with 1 negative control groups included, resulting in 8 groups total. The cRGD-DEXO/ORI concentration at which the LO2 cell survival rate was ≥90% was selected for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1\u003c/strong\u003e\u003cstrong\u003e0 Effects of cRGD-EXO/ORI on cells \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.10.1 Cell proliferation experiment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe cellular state and confluency of HCT-116 cells were observed under an inverted fluorescence microscope. When the cell reached 80% confluency, cell passage and seeding were performed. HCT-116 cells were seeded into 96-well plates at a density of 1×10\u003csup\u003e5\u003c/sup\u003e cells /mL, with 200µL cell suspension added to each well. When the cell confluency reached approximately 70%, drug intervention was performed. The old medium was discarded, 200 µL PBS solution, 6.25 µg/mL ORI, 21.6 µg/mL DEXO, 21.6 µg/mL DEXO/ORI, 21.6 µg/mL cRGD-DEXO/ORI and 3 µg/mL 5-FU solution were added to each well, respectively. The plates were then incubated in CO\u003csub\u003e2\u003c/sub\u003e incubator at 37℃ for 24 h. After incubation, 20 µL of MTT solution was added to each well, followed by incubation in the dark for 4 h. The old medium was discarded and 150 µL of DMSO solution was added to each well, with gentle shaking for 10 min. The absorbance was measured at the wavelength of 490 nm, and the data were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.10.2 Cell apoptosis was detected by acridine orange-ethidine bromide staining\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAcridine orange dye solution (reagent A), ethidine bromide dye solution (reagent B) and dilution buffer solution (reagent C) were mixed and diluted at a ratio of A:B:C=1:1:8 to prepare the working AO/EB staining solution. HCT-116 cells were digested using conventional method, then centrifuged at 1000 rpm for 5 min. The cell precipitate was collected, washed twice with PBS, and resuspended in dilution buffer (reagent C). The cell density was adjusted to (0.5~5)×10\u003csup\u003e6\u003c/sup\u003e cells /mL, and 25~50 µL cell suspension was used for subsequent staining. 2 µL of AO/EB staining solution was added, and the mixture was incubated at RT for 5~15min in the dark. After incubation, 5~10µL of stained cell suspension was dropped onto a glass slide, followed by observation and counting under a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.10.3 cell scratch test\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe horizontal lines were evenly drawn on the back of the 6-well plates with a ruler-guided marker pen (≥5 lines/well). HCT-116 cells were starved in serum-free medium for 12 h to eliminate serum interference. At 80% confluency, cells were washed 3 times with PBS, digested with trypsin, centrifugation post-termination with complete medium; supernatant removed, and the pellet was washed twice with PBS. The cells were resuspended in serum-free medium and seeded into pre-marked 6-well plates at 1×10\u003csup\u003e6\u003c/sup\u003e cells/well. After full adherence, the horizontal scratches (perpendicular to pre-drawn lines) were made with a 200 µL pipette tip; wells cleaned 3 times with PBS to remove detached cells. PBS solution, DEXO, ORI, DEXO/ORI, cRGD-DEXO/ORI and 5-FU solution were added to respective wells. Images were taken at 0 and 24 h post-treatment (3 random fields/well). Three fields of view were found, and image J software was used for analysis to calculate the mean value of relative area of intercellular migration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.11 Effects of cRGD-EXO/ORI on subcutaneous tumors in nude mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.11.1 Preparation and storage of drugs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e2g of carboxymethyl cellulose sodium (CMC-Na) was weighed and added to 398 mL of deionized double steam water. Although CMC-Na can be dissolved in both cold and hot water, it is not easy to disperse evenly when mixed directly with cold water. Thus, the powder was first pre-wetted with a small volume of hot water and stirred thoroughly to form a homogeneous paste, after which the remaining hot water was added. And then, the mixture was subjected to thorough stirring and sonication to promote rapid expansion and dissolution of the paste. Finally, the solution was left overnight to ensure complete stabilization. 5-FU was dissolved in PBS under sterile conditions and diluted to a final concentration of 30 µg/mL. D-luciferin potassium salt, a common substrate for luciferase, is widely used in the whole field of biotechnology, especially \u003cem\u003ein vivo\u003c/em\u003e imaging technology. The D-lucifcein potassium salt freeze-dried powder was first allowed to equilibrate to RT. It was then dissolved in Dulbecco's Phosphate-Buffered Saline(DPBS) to a final concentration of 15 mg/mL, followed by filtration with a 0.22 µm filter for sterilization. The resulting solution was divided into packages and stored at -20℃ in the dark.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.11.2 Establishment of transplant tumor model in BALB/c nude mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHCT-116-LUC cells are bioluminescent CRC cells that bind to the substrate D-fluoresce in potassium salt \u003cem\u003ein vivo\u003c/em\u003e, so that tumor growth can be observed in real time via small animal imaging. These cells were cultured using the same protocol as parental HCT-116 cells. Briefly, HCT-116-LUC cells in the logarithmic growth stage were collected, centrifuged, washed twice with serum-free DMEM medium, and resuspended for subsequent cell counting. The cell suspension was diluted with DMEM to a density of 2×10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003ecells/mL. BALB/c nude mice were provided by Beijing Huafukang Biotechnology Co., LTD. [SCXK (Beijing) 2019-0008] and housed in the Animal Experimental Research Center of Henan University of Chinese Medicine [SYXK (Henan) 2021-0015]. All animal procedures were approved by the Experimental Animal Ethics Committee of Henan University of Chinese Medicine (Approval No.: IACUC-202308018). Prior to injection, the skin at injection site (right axillary) of nude mice was disinfected with complex iodine. A 0.1 mL of cell suspension (containing about 2×10\u003csup\u003e6\u003c/sup\u003e cells) was injected subcutaneously into the right axillary area. To ensure uniform cell distribution, the cell suspension was thoroughly mixed before extraction. After the injection, the injection site was gently pressed with a sterile cotton swab for 1 min to prevent leakage. The growth of transplanted tumor was monitored every other day from day 2 post-tumor cell implantation. The long diameter (a) and short diameter (b) of the transplanted tumor were measured with vernier calipers, and the volume of transplanted tumor V= (a•b2)/2 was calculated. On day 7 post-implantation, the subcutaneous nodules with a diameter of 3~6 mm diameter and hard texture were visible to the naked eye at the injection site, which were identified as tumors. Tumor volumes were measured, tumor formation rate was calculated, each mouse was weighed with a balance, weight distribution were recorded and analyzed. Mice with disabilities, poor health status, or weight/tumor volume outside the range were excluded. The remaining nude mice were collectively housed in a cage, allowed to move freely for 10 min, then randomly assigned to experimental groups and numbered for documentation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.11.3 Drug administration of BALB/c nude mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe nude mice were divided into model group, ORI group, cRGD-DEXO/ORI group and 5-FU group, with 6 mice in each group. Following grouping, the volume of transplanted tumors in each nude mouse was measured again, and inter-group differences were subjected to statistically analyzed. The first day of administration was recorded as day 0. Each group was injected with 0.9% normal saline, 5 mg/kg ORI, 8.6 mg/kg cRGD-DEXO/ORI and 25 mg/kg 5-FU via tail vein every three days for a total of 6 administrations (18 days). Before each administration, the body weight of nude mice was measured using an electronic balance, and the long diameter (a) and short diameter (b) of subcutaneous tumors were measured with a vernier caliper. The graft volume V=(a·b2)/2 was calculated. To evaluate the effects of different drug treatments on the growth of transplanted colorectal cancer tumors, line graphs were plotted with time as the x-axis and tumor volume and body weight of nude mice as the y-axis respectively.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.11.4 Fluorescence detection analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLuciferase-encoding sequences are delivered to target cells or tissues via a delivery to enable luciferase expression. When corresponding substrate is introduced through intraperitoneal or intravenous injection, enzyme catalyzes luciferin oxidation to produce luminescence within a few minutes under the participation of ATP and oxygen. Notably, luminescence phenomenon occurs exclusively in living tissues, and there is a linear relationship between the intensity of luminescence and number of labeled cells. For experimental measurements, nude mice received intraperitoneal injections of 200 µL D-luciferin potassium salt on day 0 and every 3 days after post-administration, with injections administered 20 min before imaging, mice then were anesthetized with isoflurane gas and photographed using bioluminescence mode of live imaging of small animals. To analyze the data, tumor bioluminescence intensity was plotted against time (x-axis: time; y-axis: tumor bioluminescence intensity). Statistical analysis was performed on group data, with outliers (data points outside the range of mean±2 standard deviations) excluded. Tumor bioluminescence kinetic curves were then generated using the mean values of the remaining data.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.11.5 HE staining analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of dosing cycle, orbital blood extraction was used to rapidly drop blood into the 1.5 mL EP tube. The right axillary skin of nude mice was cut on the ice plate, the tumor tissue was carefully removed, weighed and photographed, and then divided into three equal parts for HE and WB detection, respectively. The heart, liver, spleen, lung, kidney and other organs were simultaneously removed for subsequent HE detection. Tumor inhibition rate of each group=(average tumor volume of control group-average tumor volume of experimental group)/average tumor volume of control group×100%.\u003c/p\u003e\n\u003cp\u003eThen tumor tissues and nude mouse organs were fixed in 10% neutral-buffered formalin for 24 h. After dehydration through graded ethanol, clearing in xylene, and infiltration with paraffin wax, samples were embedded in paraffin blocks. Sections (4~6 μm) were cut, deparaffinized, and rehydrated. Subsequently, they were stained with hematoxylin for 5~10 min, differentiated in acid alcohol, blued, and then stained with eosin for 1~2 min. After a final dehydration, clearing, and mounting, the slides were examined under a light microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.12 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental data were collected, and normality and homogeneity of variance were tested by SPSS 25.0 statistical software. If the data conform to the normal distribution and the variance is homogeneous, one-way ANOVA is used, and LSD statistical method is used for pound-by-pair comparison. The variance was not uniform, and Dunnett's T3 statistical method was used for comparison between groups. If the data does not conform to the normal distribution, the non-parametric test is used. GraphPad Prism 9 processes bar graphs, in which \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 indicated that the difference was statistically significant.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn recent years, many plant compounds have been proved to be good for disease and health, and more and more new active ingredients of Chinese medicine are effective against tumors. However, due to the poor targeting of some natural compounds, their clinical application is greatly limited. With the rise of new nanotechnology, combining these natural compounds with nanotechnology-based drug delivery systems to prepare TCM compound nanopreparations may further improve the clinical effectiveness of these drugs. Accordingly, in this study, we selected ORI a natural compound with potent anti-CRC activity and loaded it into DEXO. To improve targeting specificity, we functionalized DEXO with cRGD peptides, constructing a targeted drug delivery system (cRGD-DEXO/ORI) for CRC. Our findings confirmed that this system exhibits robust targeting to CRC cells and exerts significant anti-CRC effects both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eNotably, this study addresses two critical challenges in natural compound-based cancer therapy: poor targeting and limited bioavailability. By leveraging DEXO as a novel carrier, we not only harness the biocompatibility and natural cargo-loading capacity of exosomes but also exploit the unique advantages of \u003cem\u003eDs\u003c/em\u003e (e.g., low immunogenicity, scalable production) to overcome the limitations of conventional nanocarriers. The conjugation of cRGD further enhances tumor-specific delivery by exploiting the high expression of integrin \u0026alpha;v\u0026beta;3 in CRC, thereby minimizing off-target effects and improving therapeutic precision. Collectively, these results validate cRGD-DEXO/ORI as a viable platform to augment the anti-CRC efficacy of ORI, providing a rationale for its potential clinical application. Because ORI has water solubility, poor absorption rate and difficulty in penetrating the blood-brain barrier, we adopted DEXO to wrap ORI to overcome these shortcomings.\u003c/p\u003e\n\u003cp\u003eDBCO, as an amine reactive NHS ester, has the ability to quickly and easily attach the reactive part to virtually any primary or secondary amine group, showing its potential for high efficiency and wide range of applications. In this study, DBCO was used as an intermediate to connect cRGD and DEXO to form cRGD-DEXO under certain reaction conditions. To verify the targeting of cRGD-DEXO to HCT-116 cells, PKH67 was used to label DEXO membrane. HCT-116 cells were cultured in vitro, and DEXO and cRGD-DEXO membranes were stained with PKH67 dye. After staining, they were added to culture dishes for 24 h. The results of confocal laser microscopy showed that compared with DEXO, cRGD-DEXO showed good targeting on CRC HCT-116 cells in vitro. Interestingly, with the extension of time, cRGD-DEXO gradually transferred from cell membrane to cytoplasm and nucleus, which may be due to the fact that cRGD is a tumor penetrating peptide. It can play a \u0026quot;perforating\u0026quot; role, and then penetrate into the tumor tissue\u003csup\u003e[16]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eExosomes of different cellular origin preferentially interact with specific types of recipient cells, depending on the different classes of proteins they contain on their membranes. When exosomes bind to the surface of the recipient cell membrane, they act in two main ways. One is that proteins on the exosome membrane bind to and activate receptors on the recipient cell membrane, and the recipient cell does not take up exosomes\u003csup\u003e[17]\u003c/sup\u003e. The other is that exosomes deliver their contents into the recipient cell through direct fusion with the recipient cell membrane or endocytosis\u003csup\u003e[18]\u003c/sup\u003e. In this experiment, we played a role by targeting the delivery of cRGD-DEXO/ORI to HCT-116 cells, which bound to and internalized ORI. Studies have shown that this process is a receptor-mediated and energy-dependent active process, mainly dependent on the fossa and energy-dependent endocytosis pathways regulated by clathrin, which can be inhibited by mycotoxin and chlorpromazine\u003csup\u003e[19]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe ideal drug delivery method should not only load drugs into exosomes, but also maintain the structural and functional integrity of exosomes and drugs\u003csup\u003e[20]\u003c/sup\u003e. At present, the common drug delivery strategies include exosomal pre-delivery and post-delivery. Pre-delivery is an effective drug delivery technique, but the drug delivery efficiency of this method is low. Another drug delivery strategy is post-secretion drug delivery, which has the advantage of high drug delivery efficiency and the disadvantage of damaging the integrity of exosome membrane\u003csup\u003e[21]\u003c/sup\u003e. After comprehensive evaluation, we choose electroporation method for ORI loading. In order to explore the loading rate and encapsulation rate of ORI and cRGD-DEXO at different ratios, ORI was loaded at 300v and 250\u0026micro;F according to the literature\u003csup\u003e[22]\u003c/sup\u003e. The results showed that with the increase of ORI concentration, the encapsulation rate showed a downward trend. The reason may be that when ORI concentration was too high, the fluidity of exosome membrane would be affected, and thus the encapsulation rate would decline, or the concentration of ORI reached the upper limit of the capacity that the exosome could sustain. In addition, when the \u0026quot;truck\u0026quot; concentration of exosomes increased, the drug loading increased sharply, but the encapsulation rate decreased slightly, resulting in a decrease in the utilization of exosomes. After comprehensive consideration, the ratio of ORI to cRGD-DEXO was 1:1 for subsequent experiments, and the loading rate and encapsulation rate were 22% and 29%, respectively. In addition, we also discussed the 1:1 ratio of ORI and cRGD-DEXO, different voltage capacitance conditions (voltage: 50, 100, 250, 300, 400 and 500v, capacitance: 125, 250 and 500\u0026micro;F) on ORI loading rate and packet rate, the results confirm that ORI loading rate and packet rate reach the best when the ratio of ORI to cRGD-DEXO is 1:1 at 300v and 250\u0026micro;F. In addition, after coupling cRGD and loading ORI, the DEXO particle size increases and is well distributed, and the potential decreases, which may be related to the negative charge of cRGD itself.\u003c/p\u003e\n\u003cp\u003eIn vitro experiments in this study confirmed that cRGD-DEXO/ORI had a good inhibitory effect on HCT-116 cells, and the results of cell proliferation and AO-EB staining showed that compared with the ORI group, the cell inhibition rate and apoptosis rate in the DEXO/ORI group were significantly increased. In addition, compared with DEXO/ORI group, The inhibition rate and apoptosis rate of cRGD-DEXO/ORI group were also significantly increased. The mechanism is as follows: On the one hand, ORI coated with DEXO greatly improves ORI\u0026apos;s blood circulation time and ability to cross the blood-brain barrier. On the other hand, cRGD-DEXO/ORI can be well targeted to HCT-116 cells, which may be attributed to the high affinity of cRGD for integrin \u0026alpha;v\u0026beta;3. cRGD significantly increased the endocytosis efficiency of HCT-116 cells and increased the local effective concentration of HCT-116 cells. In vivo results showed that cRGD-DEXO/ORI also showed a good inhibitory effect on tumor growth, and had no effect on body weight in nude mice. In order to track the distribution and metabolism of exosomes in vivo, we will conduct dynamic tracing of exosomes in the following experiments.\u003c/p\u003e"},{"header":"5. Summary and prospect","content":"\u003cp\u003eIn this study, we successfully constructed a cRGD-DEXO/ORI drug delivery system targeting the colorectal site, and results demonstrated that cRGD-DEXO/ORI system has a better targeting ability to CRC cells both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e,which could be used as a potential effective therapeutic agent for cancer in future. However, there are still some shortcomings in this study. For example, we will need to verify the targeting of cRGD-DEXO/ORI \u003cem\u003ein vivo\u003c/em\u003e. On the one hand, the production of exosomes is small, the extraction time is long, the extraction steps are complicated, and the scale and engineering extraction process are lacking, which greatly limits the application of exosomes. On the other hand, the drug loading of exosomes is low, and the drug loading method needs to be further explored. At the same time, exosomes of different cell origin carry different membrane proteins on their membrane surface, which gives them different biological characteristics. How to effectively select and purify exosomes is both an opportunity and a challenge for us. The commercialization of exosomes still has a long way to go, which requires more clinically relevant systematic evaluation and comprehensive systematic comparison with protocols such as liposomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors took part in writing, reviewing, and editing the manuscript. All authors reviewed the manuscript and approved it for publication. Baiyan Wang and Shuying Feng main contributed to research conception and design; Lei Wang, Chunguang Zhou and Zilong Wang performed all experiments. Aifang Li, Shuxuan Li and Yalan Li directed the project, performed statistical analysis, and interpreted data.Yujing Huangfu and Wei Chen prepared the figures, Shuying Feng contributed to the manuscript revision, and read and approved the final submitted version. The corresponding author Shuying Feng takes primary responsibility for communication with the journal and editorial office during the submission process, throughout peer review, and during publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEth\u003c/strong\u003e\u003cstrong\u003eical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the National Natural Science Foundation of China (82402600), the Joint Funds of Science and Technology Research and Development Project of Henan Province (No. 232301420070), the basic research project of key scientific research projects of universities in Henan Province (No. 23ZX005), the Natural Science Foundation of Henan Province (232300421164), and the Key Research and Development Special Project of Henan Province (241111311200).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eYang W, Zheng H, Lv W, et al. 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Cells. 2023; 12(10): 1416.\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":"Colorectal cancer, Drug delivery system, Dunaliella salina exosomes, Oridonin, Targeting delivery","lastPublishedDoi":"10.21203/rs.3.rs-8418021/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8418021/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSome Chinese medicine monomers are limited in clinical application due to poor targeting and\u003cem\u003ein vivo\u003c/em\u003e stability, strong hydrophobicity, low bioavailability, short half-life, and systemic toxicity within the therapeutic dose range, etc. Combining these monomers with nanotechnology-based drug delivery systems can further improve the targeting and clinical efficacy of these drugs. Thus, this study, constructed a drug delivery vector targeting colorectal cancer (CRC), i.e. cRGD-DEXO with cRGD-modified exosomes of \u003cem\u003eDunaliella salina\u003c/em\u003e (DEXO), and their effects of targeting and anti-CRC were investigated at the cellular level \u003cem\u003ein vitro\u003c/em\u003e and in animal model \u003cem\u003ein vivo\u003c/em\u003e. The results verified that the anti-CRC effect is cRGD-DEXO/ORI\u0026gt;DEXO/ORI\u0026gt;ORI \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003eIn vivo\u003c/em\u003e studies showed that anti-tumor effect of CRC in nude mice was cRGD-DEXO/ORI\u0026gt;ORI. In addition, compared with non-cRGD-modified DEXO, cRGD-modified DEXO showed stronger targeting to HCT-116 cells. Furthermore, cRGD-DEXO/ORI showed significantly higher accumulation in nude mouse tumor tissues than non-cRGD-modified DEXO/ORI and free ORI, directly enhancing its in vivo anti-tumor activity by concentrating ORI at tumor sites. Summarily, our study proved that we have successfully constructed a drug delivery system (i.e. cRGD-DEXO/ORI) that targets CRC, and it exhabits a better anti-CRC activity both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. It provided a promising targeted delivery strategy for hydrophobic Chinese medicine monomers like ORI, laying a solid experimental foundation for their potential clinical translation and offering new insights into the application of nanotechnology in optimizing the therapeutic efficacy of traditional Chinese medicine against malignant tumors.\u003c/p\u003e","manuscriptTitle":"Preparation of microalgae-derived exosomes drug delivery system loaded with oridonin and evaluation of anti-colorectal cancer effect","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 08:00:06","doi":"10.21203/rs.3.rs-8418021/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-06T10:29:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-06T09:09:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-17T07:14:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332639212966096578764710920363985459134","date":"2026-01-29T15:33:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54120211759937154821083001649767475278","date":"2026-01-27T12:18:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-08T20:45:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-24T10:50:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-24T10:47:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Nanotechnology","date":"2025-12-21T14:36:07+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":"89a0eb55-193f-446b-b06b-70e9fc4ec2ba","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-25T02:53:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 08:00:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8418021","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8418021","identity":"rs-8418021","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}