Nano-Emulsification of PTX and BEZ235 inhibit colon cancer growth

Nano-Emulsification of PTX and BEZ235 inhibit colon cancer growth | 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 Article Nano-Emulsification of PTX and BEZ235 inhibit colon cancer growth Xingyao Zhu, Ruiqi Zhang, Yuhua Liu, Caiyun Yang, Qi Ding, Wenyi Gu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5424111/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 May, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract As the most common malignant tumor, colorectal cancer is a serious threat to people's health, especially the problem of drug resistance, is a huge challenge. In previous studies, we have found synergistic anti-colon cancer effects with the combination of paclitaxel and BEZ235, especially for drug-resistant colorectal cancer with excellent anti-tumor effects. Therefore, this study aims to investigate the preparation of Nano-Emulsification of paclitaxel and BEZ235 (NEs-PTX-BEZ235), and to explore the potential key parameters of its clinical transformation. To provide a new therapeutic strategy for colorectal cancer, especially for drug-resistant colorectal cancer. The nanomaterials were prepared by low energy self-emulsification method, and analyzed by Malvern laser particle size analyzer. Cell drug uptake was observed under fluorescence microscope. CCK8, Western blot and flow cytometry were used to compare the anti-cancer effects and mechanisms of different experimental groups on ordinary and drug-resistant colon cancer cells. Human colon cancer primary cells were extracted to verify the anti-tumor effect of drugs. Finally, we found that NEs-PTX-BEZ235 significantly killed colon cancer cells, especially drug-resistant cells, and better than Nab-PTX. It may play a synergistic role in reducing drug resistance, inhibiting tumor stemness and inducing apoptosis of colon cancer cells by inhibiting drug resistance proteins. Biological sciences/Cancer/Cancer therapy Biological sciences/Drug discovery colon cancer Paclitaxel BEZ235 Nano-Emulsification Anti-tumor drug resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction BEZ235 is a newly discovered inhibitor of PI3K/Akt/mTOR signaling pathway [1], which is well tolerated and has good research value as a new anti-tumor drug. On the other hand, BEZ235 has a weak ability to induce apoptosis, and can trigger cell protective autophagy to offset its anti-tumor effect, so the anti-tumor effect of single drug is not ideal [2]. Paclitaxel (PTX) is a diterpenoid alkaloid extracted from Taxus plants [3], which can promote the apoptosis of tumor cells, inhibit the division and proliferation of tumor cells [4], and will not produce cross-resistance with a variety of drugs, while it is effective against multi-drug-resistant tumors. It has been applied to the treatment of malignant tumors such as breast cancer, ovarian cancer and pancreatic cancer [5,6]. Previously, we have shown that PTX and BEZ235 have synergistic inhibitory effects on the growth of colon cancer cells and their drug-resistant cells. However, the combination of nanoemulsion loaded with PTX and BEZ235 showed more significant anti-tumor effect [7,8]. On this basis, according to the existing optimal compatibility ratio of PTX and BEZ235, we designed a Nano-Emulsification of paclitaxel and BEZ235 (NEs-PTX-BEZ235) with high efficacy, low toxicity and high patient compliance. In this study, a low energy self-emulsification method was used to prepare a Nano-Emulsification of paclitaxel and BEZ235. This emulsifier has uniform particle size distribution, stable properties, and can be fully absorbed by tumor cells. In vitro anti-tumor experiments show that this emulsifier has good effects of synergistically inhibiting tumor proliferation, promoting apoptosis, reducing tumor drug resistance and reversing tumor cell stemness, and provides a promising anti-tumor strategy for future clinical treatment of colon cancer, especially drug-resistant colon cancer. 2. Materials and methods 2.1 Materials Oxaliplatin-resistant (HCT116-LOHP) and cisplatin-resistant (HT29-DDP) human colon cancer cell lines were purchased from Shanghai Aurora Technology Co., LTD. HCT116 and HT29 human colon cancer cell lines were purchased from Shanghai Fuxiang Biotechnology Co., LTD. Cisplatin and oxaliplatin, used to maintain drug resistance in two colon cancers, were purchased from Sigma in the United States. BEZ235 and PTX were purchased from Selleck Chemical, USA. Oxaliplatin and cisplatin are dissolved in saline, and PTX and BEZ235 are stored in dimethyl sulfoxide. Sources of other reagents related to cell culture are as follows: DMEM medium (Invitgen, USA), 0.25% EDTA trypsin (Solarbio, Beijing, China), fetal bovine serum (Biolnd, Israel), penicillin-streptomycin mixture (Solarbio, Beijing, China), dimethyl sulfoxide (Solarbio, Beijing, China), PBS (Shengong, Shanghai, China). CCK-8 cell proliferation toxicity assay kit (Tong Ren Chemical Research Institute, Japan) for cell proliferation, Nile Red (Solarbio, Beijing, China), FITC-labeled anti-rabbit IgG (ABCAM, USA), cell cycle assay kit (purchased from Unitech Biotechnology, China). Western blotting detected the antibody, and its concentration was PI3K (1:1000, Abcam, USA), p-Akt (1:2000, Abcam, USA), CD44 (1:2000, Abcam, USA), CD133 (1:1000, Abcam, USA), respectively. USA), Pgp(1:1000, Cst, USA), ABCC1(1:1000, Cst, USA), β-actin (1:1000, Abcam, USA); Hanks Buffer, Type Ⅳ collagenase, Tween 20 and propylene glycol were all purchased from Beijing Solarbio, China, Capryol 90 (Gattefosse, France). 2.2 Synthesis of NEs-PTX-BEZ235 PTX and BEZ235 were dissolved in DMSO, and Tween 20 and propylene glycol were first mixed into the emulsifier by vortex shaking (where the volume ratio of Tween 20 to propylene glycol was 2:1 and shaken for about 3–5 min); then the double drug (PTX and BEZ235 were added in different concentration ratios of 1:1, 1:2, 1:4, 1:8 and 1:10) was added with Capryol 90 was added to the emulsifier and vortex shaken for 5 min; finally, the above mixture was slowly added to double distilled water and whirlpool shaken for 5 min (volume ratio of Capryol 90, emulsifier and water was 20:40:40) to form the nano complex formulation. The average particle size and stability of the nano complexed formulations (1:1 ~ 1:10) were examined by a Malvern laser particle size analyzer, and the prepared nano complexed formulations were stored at 4°C. 2.3 CCK-8 cell viability determination The four cell lines were cultured in DMEM medium containing 10% fetal bovine serum and 5% penicillin/streptomycin. The HT29DDP cell-resistant line and the HCT116-LOHP cell-resistant line were supplemented with cisplatin (1.5 µg/mL) and oxaliplatin (10 µg/mL), respectively, in the medium to maintain the corresponding resistant phenotypes of the two drug-resistant cells. Cells were inoculated on 96-well plates at a cell density of 4×103 cells/well. After 24 hours, the original DMEM medium was removed and replaced with fresh DMEM medium containing different concentrations of BEZ235, PTX, PTX + BEZ235, NE-PTX, NE-PTX combined with BEZ235, NEs-PTX-BEZ235 (1:1, 1:2, 1:4, 1:8 and 1:10 groups) and albumin-paclitaxel (Nab-PTX), respectively. Among them, general cell lines were mixed with PTX or NE-PTX medium of different concentrations using 25 nm BEZ235. In contrast, drug-resistant cell lines were mixed with PTX or NE-PTX medium of different concentrations using 50 nm BEZ235 on 96-well plates. After 48h of treatment, 10µL CCK-8 reagent was added to each well under light protection, and the 96-well plate was wrapped in aluminum foil and placed in the incubator. Two hours later, the absorbance OD value was measured at A450 nm using the BIO-RAD xMark microplate instrument, and the cell survival rate was calculated according to the following formula: Cell survival rate% = (absorbance OD value of experimental group-absorbance of blank group) ÷ (absorbance OD value of control group-absorbance of blank group) × 100% A combination index was calculated to determine the synergetic effect of combination treatment based on the following formula: CI = \(\:\frac{\text{a}\text{v}\text{a}\text{i}\text{l}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}\:\text{o}\text{f}\:\text{B}\text{E}\text{Z}235}{\text{C}\text{e}\text{l}\text{l}\:\text{a}\text{v}\text{a}\text{i}\text{l}\text{a}\text{b}\text{i}\text{t}\text{y}\:\text{o}\text{f}\:\text{N}\text{E}-\text{P}\text{T}\text{X}\times\:\:\text{c}\text{e}\text{l}\text{l}\:\text{a}\text{v}\text{a}\text{i}\text{l}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}\:\text{o}\text{f}\:\text{B}\text{E}\text{Z}235}\) CI 1 indicated synergic, additive and antagonistic effect, respectively 2.4 In vitro cellular uptake of drugs NE-Nile red was prepared by encapsulating Nile red dye in nanoemulsion. Cells were seeded in 6-well plates at a cell density of 2 × 10 5 cells/well. After 24 hours, the original medium was removed and replaced by the medium containing NE-Nile red. After 0, 12, 24 and 48 hours of incubation, the plates were washed with PBS, fixed with paraformaldehyde for 15 minutes, washed with PBS again, and incubated with DAPI for 15 minutes to stain the nuclei. Then the uptake of NE-Nile red by tumor cells was observed under a fluorescence microscope. 2.5 The expression of multidrug resistance, PI3K/Akt/mTOR pathway related molecules and dry protein were detected by Western blot. The total protein was extracted after the cells were treated with different drugs for 48 hours. The protein concentration was measured with a nanodrop spectrophotometer. Proteins were separated by Bio-RAD gel electrophoresis and transferred to PVDF membrane. After blocking for 2 H at room temperature (5% bovine serum albumin), the cells were incubated with rabbit anti-human Pgp, ABCC1, PI3K, p-AKt, CD133, CD44 and mouse anti-human β-actin antibodies overnight at 4 ℃, washed at room temperature, and treated with horseradish peroxidase-conjugated anti-rabbit and mouse antibodies for 2 H, then observed and imaged by ECL. 2.6 Flow cytometry for cell cycle detection experiments 2 * 10 5 -1 * 10 6 cells were collected, washed once with PBS, and centrifuged to discard the supernatant. Precooled 80% ethanol at -20.degree. C. was added and fixed overnight. On the day of detection, the fixed cells were centrifuged, the supernatant was discarded, washed with PBS, added with 1 ml DNA Staining solution, vortexed for 5–10 seconds, and incubated for 30 minutes at room temperature in the dark. Detection was performed on a flow cytometer. 2.7 Colon cancer primary cell extraction Primary cells were cultured by tissue block attachment method and collagenase digestion method. The fresh tissues of human colorectal cancer were collected and stored in the preservation solution (45ml DMEM + 5ml penicillin-streptomycin mixture). The tissues were taken out from the ultra-clean table, fat and necrotic tissues were removed, and washed with Hank's solution. The washed tissues were cut into small pieces of 1mm 3 , washed with Hank's solution again, and the supernatant was discarded. It was resuspended with preservation solution and evenly arranged at the bottom of the culture flask. After adding DMEM culture medium, the culture flask was placed with the bottom upward for 2–4 hours. When there was cell growth around the tissue block, it was considered that the culture was successful. Collagenase digestion method: The tissue block was cut into small pieces of 1 mm 3 , washed with Hanks solution, added with type IV collagenase, and placed in a 37 ℃ water bath for 1 H until the tissue block was dispersed, and then the cell suspension was collected and filtered through a 200-mesh sterile cell filter. The liquid that passed through the screen was transferred to a flask that had been previously filled with DMEM medium. Cells were cultured for 2–3 weeks and then purified by trypsin digestion. 2.8 Statistical analysis The data were analyzed by SPSS.20 statistical analysis software, and the results were presented in the form of mean ± standard deviation. The t-test was used to compare pairwise samples, and the analysis of variance was used to compare multiple samples. The difference was considered statistically significant at P < 0.05. 3. Results 3.1 Characterization of NEs-PTX-BEZ235 The ratios of PTX to BEZ235 in the compound were set at 1:1, 1:2, 1:4, 1:8 and 1:10. The Malvern laser particle size analyzer examined the nanoparticle size (Fig. 1 A), and the results showed that the particle size distribution of the nano-emulsion (NE) system was narrow, with an average particle size of 171.1 nm and a polydispersion index (PDI) of 0.142–0.202. The particle size and PDI were similar in alkaline DMEM medium, neutral PBS and pH5.8 buffer. During the subsequent six days of continuous measurements (Fig. 1 B), we found that the distribution and particle size of NEs-PTX-BEZ235 remained in the same range without significant changes, especially in the DMEM medium, indicating good stability of NEs-PTX-BEZ235. 3.2 In vitro cellular uptake of drugs NE-Nile red was prepared by encapsulating Nile red dye in nanoemulsion. In colon cancer cells HT29, HCT116 and colon cancer drug-resistant cells HT29-DDP, HCT116-LOHP, the uptake of NE-Nile red by tumor cells at four time points of 0, 12, 24 and 48 hours was observed. The results showed that NE-Nile red was gradually absorbed and ingested by colon cancer cells and drug-resistant cells with the increase of time. It was almost completely absorbed into the cytoplasm of tumor cells at 48 H (Fig. 2 ), indicating that there was a good uptake of NE by colon cancer cells. 3.3 Inhibitory effects on tumor cells of single drug group, double drug combination group, nano single drug group, NE-PTX combined with BEZ235 group, NEs-PTX-BEZ235 group and albumin-paclitaxel (Nab-PTX) group Under the same drug concentration, the effect of the compound preparation 1:10 group was better than that of the other experimental groups, and its IC50 was 2.096 and 1.276 in HT29 and HCT116 cells, respectively (Fig. 3 A). 9.836 and 6.803 in the drug-resistant cell lines HT29-DDP and HCT116-LOHP, respectively (Fig. 3 B). The combination index (CI) of NEs-PTX-BEZ235 (1:10) was calculated, demonstrating the synergistic effect of PTX and BEZ235 in the compound preparation. 3.4 NEs-PTX-BEZ235 can significantly reduce the expression of PI3K/Akt/mTOR pathway related proteins p-AKt and PI3K in colon cancer cells and drug-resistant cells. BEZ235 is an inhibitor of PI3K/Akt/mTOR signaling pathway, and blocking PI3K/AKT signaling pathway can enhance the drug sensitivity of drug-resistant human colon cancer cells [1,9]. In this study, Weston blot was used to detect and compare the effects of different drug groups on the expression of PI3K and p-Akt in colon cancer cells and drug-resistant cells. As a result of detection in colon canc cells HT29, HCT116, HT29-DDP, and HCT116-LOHP (Fig. 4 A-B), The expressions of PI3K and p-Akt in the BEZ235 single drug group, PTX single drug group, PTX + BEZ235 group, NE-PTX group, NE-PTX combined with BEZ235 group and the NEs-PTX-BEZ235 groups with different double drug ratios are all down-regulated compared with the negative control group, and the down-regulation effect of the NEs-1: 10 group is the most obvious. The difference was statistically significant (P < 0.05), indicating that the inhibitory effect of NEs-1: 10 group was better than that of other experimental groups. 3.5 NEs-PTX-BEZ235 preparation can significantly reduce the expression of CD44 and CD133 in colon cancer cells. BEZ235 can inhibit the growth of colon cancer stem cells [1,10]. In order to further explore the possible mechanism of tumor inhibition by drugs, Weston blot was used to detect and compare the effects of different drug groups on the expression of stemness related proteins CD133 and CD44 in colon cancer cells and drug-resistant cells. As a result, in the colon cancer cells HT29, HCT116, HT29-DDP, and HCT116-LOHP (Fig. 4 C-D). Compared with the negative control group, the expressions of CD133 and CD44 in the BEZ235 single drug group, PTX single drug group, PTX combined with BEZ235 group, NE-PTX group, NE-PTX combined with BEZ235 group and NEs-PTX-BEZ235 groups with different double drug ratios were all down-regulated, and the down-regulation effect of the NEs-1: 10 group was the most obvious. The difference was statistically significant (P < 0.05), indicating that the inhibitory effect of NEs-1: 10 group was better than that of other experimental groups. 3.6 NEs-PTX-BEZ235 can significantly reduce the expression of Pgp and ABCC1 protein in drug-resistant cells. Chemotherapy for colon cancer often leads to the mutation of some genes in the tumor, or stimulates normal cells to produce some chemical factors suitable for tumor growth, thus causing multidrug resistance [11]. In order to explore the mechanism of PTX and BEZ235 on drug-resistant colon cancer cells, we compared the expression of P-glycoprotein (Pgp) and multidrug resistance-associated protein (ABCC1) in different treatment groups by Western blotting. It was found that in HT29DDP and HCT116-LOHP (Fig. 4 E), Compared with the negative control group, the expressions of Pgp and ABCC1 in the BEZ235 single drug group, PTX single drug group, PTX combined with BEZ235 group, NE-PTX group, NE-PTX combined with BEZ235 group and NEs-PTX-BEZ235 groups with different double drug ratios are all down-regulated, and the down-regulation effect of the NEs-1: 10 group is the most obvious. The difference was statistically significant (P < 0.05), indicating that the inhibitory effect of NEs-1: 10 group was better than that of other experimental groups. 3.7 Flow cytometry was used to analyze and compare the effects of different proportions of NEs-PTX-BEZ235 on the cell cycle of colon cancer cells and their drug-resistant cells. In the flow cytometry results of colon cancer cells HT29 and HCT116 (Fig. 5 A-B) and colon cancer drug-resistant cells HT29-DDP (Figure) and HCT116-LOHP (Fig. 5 C-D), it was found that, compared with the untreated control group, NEs-PTX-BEZ235 groups could induce G2/M phase arrest of cells, thereby promoting apoptosis of tumor cells. Among them, the NEs-1: 10 group had the best apoptosis-promoting effect, and the difference was statistically significant (P < 0.05) (Fig. 5 E), and the effect was better at high concentration, which also indicated that the induction of G2/M phase arrest by nano-compound drugs on colon cancer cells and their drug-resistant cells was concentration-dependent. 3.8 NEs-PTX-BEZ235 has significant inhibitory effect on primary colon cancer cells The primary cells of colorectal cancer were extracted from human colorectal cancer specimens and purified by trypsin digestion after successful culture, and the extracted cells were tested by flow cytometry. The CCK-8 method was used to detect the primary colon cancer cells treated with the gradient concentration of NEs-PTX-BEZ235, and the results showed that in the primary colon cancer cells, after 48 hours of drug administration, under the same drug concentration, the survival rate of tumor cells in the NEs-1:10 group decreased more significantly (Fig. 6 ). The IC50 values of PTX/BEZ235 nanocomposites (1:1, 1:2, 1:4, 1:8 and 1:10) were 5. 371 nM, 3.813 nM, 4.475 nM, 3.857 nM and 3. 471 nM, respectively. 4. Discussion PTX is considered to be one of the most successful natural anticancer drugs [12], which can promote the assembly of tubulin into microtubules, prevent the dissociation of microtubules, and induce G2/M arrest of colon cancer cells, thereby blocking cell cycle progression, preventing mitosis, inducing the activation of caspase8 apoptosis pathway, and inhibiting cancer cell growth [4,13]. However, PTX has low solubility in water, many side effects, and no targeting [14], which greatly limits its clinical application. BEZ235 is a dual inhibitor of PI3K/Akt/mTOR signaling pathway, which down-regulates the expression of ATP-binding transporters (such as Pgp), reverses Pgp-mediated multidrug resistance and promotes apoptosis. It has been reported that BEZ235 can enhance the sensitivity of colon cancer cell line HCT-116 to 5-fu and inhibit the growth of colon cancer stem cells [1,9]. Combination therapy is a common strategy to overcome drug resistance. Therefore, we speculate that BEZ235 and PTX may act synergistically on multiple targets of colon cancer cells, thereby reducing drug resistance and further promoting apoptosis. Nanoemulsion is a stable, transparent, low-viscosity, uniform and thermodynamically stable dispersion system composed of oil phase, water phase, surfactant and cosurfactant in a certain proportion, which can increase the solubility of poorly soluble drugs and improve the stability and bioavailability of drugs [22,23]. In our previous studies, it has been proved that the combination of PTX and BEZ235 has a good synergistic inhibitory effect on the growth of colon cancer cells with K-ras mutation +/-and their drug-resistant cells, and the application of nanoemulsion loaded with PTX combined with low concentration of BEZ235 can further enhance the efficacy [8]. The purpose of this study is to complete the design and development of NEs-PTX-BEZ235 compound preparation according to the optimal proportion, verify its stability, explore its anti-tumor effect and mechanism of action, and verify it in clinical samples, so as to lay a foundation for exploring its potential clinical application value. Firstly, PTX and BEZ235 were wrapped into nanoemulsion according to different concentration ratios of 1: 1,1: 2,1: 4,1: 8 and 1:10, referring to the previous preparation method of paclitaxel loaded nanoemulsion, and the nanoparticle size of compound preparation was detected by Malvern laser particle size analyzer. The results showed that the particle size of NEs-PTX-BEZ235 was uniformly distributed in alkaline DMEM medium, neutral PBS and acidic buffer (pH 5.8), the particle size range was 100–200 nM, the average particle size was 171.1 nM, and the polydispersity index (PDI) was 0.142–0.202 (< 0.3). Indicating that the particle size distribution is relatively concentrated. In the subsequent 6 days of continuous measurement, we found that the particle size distribution of NEs-PTX-BEZ235 did not change significantly, especially in DMEM medium, which indicated that NEs-PTX-BEZ235 had good stability. After that, we detected the uptake of empty nanoemulsion (NE) by tumor cells in vitro by fluorescence experiment, and found that HT29, HCT116, HT29-DDP and HCT116-LOHP cells had good uptake of NE. Then, we compared the effects of single drug group, double drug combination group, nano single drug group, nano paclitaxel combined with BEZ235 group, NEs-PTX-BEZ235 group and albumin-paclitaxel (Nab-PTX) group on the proliferation of colon cancer HT29, HCT116, HT29-DDP and HCT116-LOHP cells through CCK8 experiment. And calculate that survival rate of the tumor cells. The results showed that both PTX and BEZ235 could inhibit the proliferation of colon cancer cells and their drug-resistant cells in a dose-dependent manner, but when BEZ235 was used alone, the concentration was too high, and the inhibitory effect on drug-resistant colon cancer cells was not obvious. The IC50 of HT29, HCT116, HT29-DDP and HCT116-LOHP cells in the nanoemulsion loaded with PTX or BEZ235 was significantly lower than that in the single drug group, and the difference was statistically significant (P < 0.05), indicating that after the nanoemulsion was loaded with PTX or BEZ235, The dosage can be reduced; Colon cancer cell lines HT29 and HCT116 and their drug-resistant cell lines HT29-DDP and HCT116-LOHP were treated with gradient concentrations of PTX or NE-PTX combined with low concentrations of BEZ235 in dual-drug combination therapy. The results showed that with the increase of PTX or NE-PTX concentration, The IC50 of tumor cells decreased more significantly than that of single drug group, suggesting that the combination of NE-PTX and BEZ235 can complement each other and enhance each other's sensitivity. When NE-PTX combined with low concentration of BEZ235, it showed more intense tumor inhibition. In that proces of treating colon canc cell strains HT29 and HCT116 and drug-resistant cell strains HT29-DDP and HCT116-LOHP thereof, the nano paclitaxel-BEZ235 compound preparation with gradient concentration and different double-drug matching is apply, When the concentration ratio of NE-PTX and BEZ235 was 1:10, the effect was the best. The IC50 of PTX and BEZ235 in normal colon cancer cell lines was reduced to about 1/12 of that of paclitaxel alone, and the dosage in drug-resistant cell lines was also reduced to about 2/5 of that of paclitaxel alone. The effect was better than that of NE-PTX combined with BEZ235. In addition, the IC50 of the NEs-PTX-BEZ235 was significantly lower than that of albumin-paclitaxel (Nab-PTX), which is currently used in clinical practice. PI3K/Akt/mTOR signaling pathway is closely related to multidrug resistance of tumors, and plays an important role in the occurrence and development of tumors, which is considered as a new target for chemotherapy resistance treatment [15,16]. The combination of PTX and BEZ235 could effectively inhibit the expression of PI3K/Akt/mTOR pathway-related proteins PI3K and p-Akt, and the inhibitory effect was more obvious when it was prepared into NEs-PTX-BEZ235, and the effect of NES-1: 10 group was the best, with statistically significant difference (P < 0.05). There are some special tumor cell subsets in the tumor cell population, which are called cancer stem cells (CSCs) or tumor initiating cells (TICs) [17,18]. These cells have malignant biological potentials such as self-renewal, multi-directional differentiation, unlimited proliferation and tumor reconstruction, and are considered to be the root of tumorigenesis, progression, invasion and metastasis, chemoradiotherapy resistance and recurrence [19,20]. CD44 and CD133 can be used as markers of colon cancer stem cells. Through Western blotting experiments, we found that compared with other experimental groups, The inhibition of CD44 and CD133 expression in colon cancer cells HT29, HCT116 and their drug-resistant cells HT29-DDP, HCT116-LOHP by NEs-1:10 group was the best, and the difference was statistically significant (P < 0.05). ABCC1/MRP1 and ABCB1/Pgp are energy-dependent drug efflux pumps with an ATP-binding site and a drug-binding site. ATP can be hydrolyzed by binding to ABCC1 and Pgp to provide energy. This energy is used to pump out the drug entering the cell, resulting in the failure of the intracellular drug concentration to reach an effective level, thus causing drug resistance and leading to chemotherapy failure [8,21]. Our previous study demonstrated that the combination of PTX and BEZ235 can effectively inhibit the expression of drug resistance proteins ABCC1 and Pgp in colon cancer cells [8]. In this study, we also proved that the NEs-PTX-BEZ235 could down-regulate the expression of ABCC1 and Pgp in drug-resistant colon cancer cell lines, and the down-regulation effect of NEs-1: 10 group was the most obvious, and the difference was statistically significant (P < 0.05). PTX can induce G2/M phase arrest and activation of caspase8 apoptosis pathway in colon cancer cells [13]. NEs-PTX-BEZ235 can also induce G2/M arrest of colon cancer cells HT29, HCT116 and colon cancer drug-resistant cells HT29-DDP, HCT116-LOHP, thus inhibiting the growth of tumor cells, and the NEs-1: 10 group has the best effect. To sum up, our experimental results show that NEs-PTX-BEZ235 can effectively improve the drug sensitivity of colon cancer cells and drug-resistant cells, and its synergistic killing effect may be related to promoting the programmed death process of tumor cells and inhibiting the stemness and drug resistance of colon cancer cells. It was found that this emulsifier had the same obvious anti-cancer effect in human colon cancer primary cells, suggesting that the compound preparation is expected to become an effective means for the treatment of drug-resistant colon cancer. Declarations Conflict of interest The authors have no conflicts of interest to declare. Funding This research was funded by the National Natural Science Foundation of China (grant numbers 82373176) and the Zhejiang Province Basic Public Welfare Research Program Project (grant number LY23H160012). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Author Contribution Xingyao Zhu: Data curation, Formal analysis, Methodology, Validation, Writing - original draft. Ruiqi Zhang: Methodology, Resources, Writing - original draft. Yuhua Liu: Data curation. Caiyun Yang: Data curation. Qi Ding: Data curation. Wenyi Gu: Supervision. Hong Zou: Conceptualization, Formal analysis, Funding acquisition, Project administration. Data Availability All data generated or analysed during this study are included in this published article [and its supplementary information files] References Roper J, Richardson MP, Wang WV, et al. The dual PI3K/mTOR inhibitor NVP-BEZ235 induces tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer. PloS one. 2011;6:e25132. Lidi Z, Zhen L, Bin J, et al. Research progress of NVP-BEZ235 combined with anti-tumor drugs. Basic Clin Med. 2016;36:1450-4. Gang C, Qian Z, Min W, et al. Research progress of endophytic fungi producing taxol. Chinese Wild Plant Resources. 2015;34:24 − 9. Pham DT, Saelim N, Tiyaboonchai W. Paclitaxel loaded EDC-crosslinked fibroin nanoparticles: a potential approach for colon cancer treatment. 2020;10:413 − 24. Zhiwen L, Zhangjun Q. Progress in clinical application of paclitaxel liposomes. Asia-Pac Trad Med. 2012;8:229 − 32. Haitao Y. Progress in synthesis and application of anticancer drug paclitaxel. Heilongjiang Science and Technology Information. 2017:129. Zou H, Li L, Garcia Carcedo I, et al. Synergistic inhibition of colon cancer cell growth with nanoemulsion-loaded paclitaxel and PI3K/mTOR dual inhibitor BEZ235 through apoptosis. International journal of nanomedicine. 2016;11:1947-58. Hu Y, Zhang K, Zhu X, et al. Synergistic Inhibition of Drug-Resistant Colon Cancer Growth with PI3K/mTOR Dual Inhibitor BEZ235 and Nano-Emulsioned Paclitaxel via Reducing Multidrug Resistance and Promoting Apoptosis. 2021;16:2173-86. Chen YH, Wang CW, Wei MF, et al. Maintenance BEZ235 Treatment Prolongs the Therapeutic Effect of the Combination of BEZ235 and Radiotherapy for Colorectal Cancer. 2019;11. Chen J, Shao R, Li F, et al. PI3K/Akt/mTOR pathway dual inhibitor BEZ235 suppresses the stemness of colon cancer stem cells. Clinical and experimental pharmacology & physiology. 2015;42:1317-26. Zhang YK, Wang YJ, Lei ZN, et al. Regorafenib antagonizes BCRP-mediated multidrug resistance in colon cancer. Cancer letters. 2019;442:104 − 12. Weaver BA. How Taxol/paclitaxel kills cancer cells. Molecular biology of the cell. 2014;25:2677-81. Tiwari S, Tirosh B, Rubinstein A. Increasing the affinity of cationized polyacrylamide-paclitaxel nanoparticles towards colon cancer cells by a surface recognition peptide. International journal of pharmaceutics. 2017;531:281 − 91. Gonçalves A, Braguer D, Carles G, et al. Caspase-8 activation independent of CD95/CD95-L interaction during paclitaxel-induced apoptosis in human colon cancer cells (HT29-D4). Biochemical pharmacology. 2000;60:1579-84. Arnold M, Sierra MS, Laversanne M, et al. Global patterns and trends in colorectal cancer incidence and mortality. Gut. 2017;66:683 − 91. Matsuno J, Kanamaru T, Arai K, et al. Synthesis and characterization of nanoemulsion-mediated core crosslinked nanoparticles, and in vivo pharmacokinetics depending on the structural characteristics. Journal of controlled release : official journal of the Controlled Release Society. 2020;324:405 − 12. Dreesen O, Brivanlou AH. Signaling pathways in cancer and embryonic stem cells. Stem cell reviews. 2007;3:7–17. Hennessy BT, Smith DL, Ram PT, et al. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nature reviews Drug discovery. 2005;4:988–1004. Feltbower RG, Picton S, Bridges LR, et al. Epidemiology of central nervous system tumors in children and young adults (0–29 years), Yorkshire, United Kingdom. Pediatric hematology and oncology. 2004;21:647 − 60. Qiang H, Jun D, Yude Z, et al. Isolation and culture of tumor stem cells from human glioma tissue. Chinese Journal of Oncology. 2006:331-3. Bao S, Wu Q, McLendon RE, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006;444:756 − 60. Eramo A, Ricci-Vitiani L, Zeuner A, et al. Chemotherapy resistance of glioblastoma stem cells. Cell death and differentiation. 2006;13:1238-41. Wang Z, Sun X, Feng Y, et al. Dihydromyricetin reverses MRP2-induced multidrug resistance by preventing NF-κB-Nrf2 signaling in colorectal cancer cell. Phytomedicine : international journal of phytotherapy and phytopharmacology. 2021;82:153414. Additional Declarations No competing interests reported. Supplementary Files CCK1.xlsx CCK2.xlsx WBsupplement.pdf Flowcytometry.xlsx Cite Share Download PDF Status: Published Journal Publication published 09 May, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 09 Dec, 2024 Reviews received at journal 07 Dec, 2024 Reviewers agreed at journal 04 Dec, 2024 Reviews received at journal 30 Nov, 2024 Reviewers agreed at journal 30 Nov, 2024 Reviewers invited by journal 30 Nov, 2024 Editor assigned by journal 30 Nov, 2024 Editor invited by journal 14 Nov, 2024 Submission checks completed at journal 13 Nov, 2024 First submitted to journal 09 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5424111","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":387823679,"identity":"d03f58e2-c2cb-4ac3-b123-4a8082d0f1fa","order_by":0,"name":"Xingyao Zhu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Xingyao","middleName":"","lastName":"Zhu","suffix":""},{"id":387823680,"identity":"88ad2e9f-0100-407e-ba05-9126cc883346","order_by":1,"name":"Ruiqi Zhang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Ruiqi","middleName":"","lastName":"Zhang","suffix":""},{"id":387823681,"identity":"d220f6e8-9e0e-4b65-adb5-b5f24cc32684","order_by":2,"name":"Yuhua Liu","email":"","orcid":"","institution":"Shihezi University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuhua","middleName":"","lastName":"Liu","suffix":""},{"id":387823682,"identity":"e341c76b-10b5-4b20-be97-e3e9f41c4c39","order_by":3,"name":"Caiyun Yang","email":"","orcid":"","institution":"Shihezi University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Caiyun","middleName":"","lastName":"Yang","suffix":""},{"id":387823683,"identity":"82446301-c058-411a-a4c1-46ede70217d3","order_by":4,"name":"Qi Ding","email":"","orcid":"","institution":"Shihezi University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Ding","suffix":""},{"id":387823684,"identity":"a99b15be-b939-4b79-a9c5-898f883faf58","order_by":5,"name":"Wenyi Gu","email":"","orcid":"","institution":"University of Queensland","correspondingAuthor":false,"prefix":"","firstName":"Wenyi","middleName":"","lastName":"Gu","suffix":""},{"id":387823685,"identity":"ffe7a625-1760-4ab8-b3a5-79a2b6d1a506","order_by":6,"name":"Hong Zou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYHACNhAhx8BwAMYmUosx6VoSGxBsAkC+vcfswccdtenzG88YMHwoO8zAP7sBvxbGnjPmhjPPHM9tbDhjwDjj3GEGiTsH8Gthlsgxk+ZtO5bbzHDGgJm37TCDgUQCAY9AtaSzgbT8JUYLD0RLTQIPSAsjMVokeI6VSc5sO2A4g+FYwcGec+k8EjcIaJFvb94m8bGtTl5+xuGND36UWcvxzyCgBQoOA+07AI5MHqLUA0EdAwN/A7GKR8EoGAWjYKQBAPBlP9ZUBc4QAAAAAElFTkSuQmCC","orcid":"","institution":"The Second Affiliated Hospital of Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"Hong","middleName":"","lastName":"Zou","suffix":""}],"badges":[],"createdAt":"2024-11-10 03:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5424111/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5424111/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-94620-y","type":"published","date":"2025-05-09T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71266784,"identity":"1fcfbb75-6984-42fe-9a7b-7c41fff32c31","added_by":"auto","created_at":"2024-12-12 17:54:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6001952,"visible":true,"origin":"","legend":"\u003cp\u003eDLS analysis NEs-PTX-BEZ235. A. NEs-PTX-BEZ235 (1:1 ~ 1:10) has a narrow particle size distribution with a polydispersity index between 0.142 and 0.202, and has similar particle size and PDI in DMEM medium, PBS and pH 5.8 buffer; B. The particle size distribution of nanometer compound preparation (1:1 ~ 1:10) was measured for 6 consecutive days. It was found that the particle size did not change significantly in alkaline, neutral and acidic media, especially in alkaline DMEM medium.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5424111/v1/8ef6b3ee3891752c971ba13b.png"},{"id":71267783,"identity":"78faee26-8c99-45ad-9bbd-1ddafef60816","added_by":"auto","created_at":"2024-12-12 18:10:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2310201,"visible":true,"origin":"","legend":"\u003cp\u003eWith the increase of time, NE was gradually taken up by tumor cells, and almost completely entered the cells after 48 hours.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5424111/v1/26b5f3a3ff139a5692b770bb.jpg"},{"id":71267782,"identity":"b6574d87-7558-426f-95bf-2518cae6d581","added_by":"auto","created_at":"2024-12-12 18:10:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1144184,"visible":true,"origin":"","legend":"\u003cp\u003eCCK8 method was used to detect the effects of single drug group, nano single drug group, double drug combination group and NEs-PTX-BEZ235 group on the proliferation of tumor cells. A. The effect of different experimental groups on the proliferation of colon cancer cells HT29 and HCT116 was studied. The compound preparation 1:10 group had the best anti-cancer effect, and its IC50 was 2.096 and 1.276 in HT29 and HCT116 cells, respectively. B. The effect of different experimental groups on the proliferation of drug-resistant colon cancer cells HT29-DDP and HCT116-LOHP showed that the compound preparation 1:10 group had the best anti-cancer effect, and its IC50 in HT29-DDP and HCT116-LOHP cells were 9.836 and 6.803, respectively, * indicating P \u0026lt; 0.05. * * indicates P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5424111/v1/bf5b5ebf367fc77ead9d102d.jpg"},{"id":71267785,"identity":"0694108a-2f3f-4714-881a-1fe834836694","added_by":"auto","created_at":"2024-12-12 18:10:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2871189,"visible":true,"origin":"","legend":"\u003cp\u003eA-B. Effects of different experimental groups on PI3K/Akt/mTOR pathway related proteins in tumor cells. * means P \u0026lt; 0.05, * * means P \u0026lt; 0.01\u003c/p\u003e\n\u003cp\u003eC-D. Effects of Different Experimental Groups on Tumor Cell Stem Protein. * means P \u0026lt; 0.05, * * means P \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003eE. Effects of different experimental groups on drug-resistant protein of colon cancer drug-resistant cells. * means P \u0026lt; 0.05, * * means P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5424111/v1/0037de9302bfc07a687f7c6a.jpg"},{"id":71266780,"identity":"12538c0e-29d4-45fd-86be-585ce7c0aba6","added_by":"auto","created_at":"2024-12-12 17:54:25","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2583107,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry was used to analyze and compare the effects of different proportions of NEs-PTX-BEZ235 on the cell cycle of tumor cells. In HT29 (A) and HCT116 (B), HT29-DDP (C) and HCT116-LOHP (D), different compound preparations could induce G2/M phase arrest of tumor cells, and the effect of NEs-1: 10 group was the most obvious. E. Comparison of the percentage of cells in G2/M phase after treatment between different administration groups, * and * * indicate P \u0026lt; 0.05 and P \u0026lt; 0.01, respectively, compared with the control group.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5424111/v1/7dd90eb74dc28f67b45b4b35.jpg"},{"id":71267781,"identity":"2ac32c43-0954-48d1-a6de-2ba82b60caaa","added_by":"auto","created_at":"2024-12-12 18:10:25","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":309309,"visible":true,"origin":"","legend":"\u003cp\u003eThe primary colon cancer cells were treated with different proportions of NEs-PTX-BEZ235 with gradient concentration, and the effects of different groups on the proliferation of primary colon cancer cells were detected and compared. A. Extracted human colon cancer primary cells (100 ×); B. The positive rate of CK20 was 90.5% by flow cytometry. The positive rate of CDX2 was 91.7% by flow cytometry. The IC50 of different proportions of NEs-PTX-BEZ235 on primary human colon cancer cells were 5.371 nM, 3.813 nM, 4.475 nM, 3.857 nM and 3.471 nM, respectively. * indicates P \u0026lt; 0.05, * * indicates P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5424111/v1/582fa9db228fec3e4202dce0.jpg"},{"id":82537508,"identity":"3d643656-fc56-4bc5-aa52-921d8c4ea9bb","added_by":"auto","created_at":"2025-05-12 16:07:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15420928,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5424111/v1/380bc1c1-a20a-40e2-b1d1-ac66d8672e9e.pdf"},{"id":71266813,"identity":"90688da8-0411-4fcd-af41-816f73cddb2f","added_by":"auto","created_at":"2024-12-12 17:54:29","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29851,"visible":true,"origin":"","legend":"","description":"","filename":"CCK1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5424111/v1/8c36255ad7ae05a5be77c480.xlsx"},{"id":71267174,"identity":"c762cdb3-38cc-4a44-8351-b19d6ddd2802","added_by":"auto","created_at":"2024-12-12 18:02:24","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31797,"visible":true,"origin":"","legend":"","description":"","filename":"CCK2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5424111/v1/c7a70180d56ef5159c80b4f1.xlsx"},{"id":71266796,"identity":"8d82770a-9ed3-4ebd-8b44-aed876d219c5","added_by":"auto","created_at":"2024-12-12 17:54:27","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1128873,"visible":true,"origin":"","legend":"","description":"","filename":"WBsupplement.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5424111/v1/5f9083460b2d64f61c4a4953.pdf"},{"id":71267181,"identity":"986bc354-f452-4d04-8a3f-867d05ffa934","added_by":"auto","created_at":"2024-12-12 18:02:25","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13614,"visible":true,"origin":"","legend":"","description":"","filename":"Flowcytometry.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5424111/v1/78f4d5e719d391c4b3a7e727.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nano-Emulsification of PTX and BEZ235 inhibit colon cancer growth","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBEZ235 is a newly discovered inhibitor of PI3K/Akt/mTOR signaling pathway [1], which is well tolerated and has good research value as a new anti-tumor drug. On the other hand, BEZ235 has a weak ability to induce apoptosis, and can trigger cell protective autophagy to offset its anti-tumor effect, so the anti-tumor effect of single drug is not ideal [2]. Paclitaxel (PTX) is a diterpenoid alkaloid extracted from Taxus plants [3], which can promote the apoptosis of tumor cells, inhibit the division and proliferation of tumor cells [4], and will not produce cross-resistance with a variety of drugs, while it is effective against multi-drug-resistant tumors. It has been applied to the treatment of malignant tumors such as breast cancer, ovarian cancer and pancreatic cancer [5,6].\u003c/p\u003e \u003cp\u003ePreviously, we have shown that PTX and BEZ235 have synergistic inhibitory effects on the growth of colon cancer cells and their drug-resistant cells. However, the combination of nanoemulsion loaded with PTX and BEZ235 showed more significant anti-tumor effect [7,8]. On this basis, according to the existing optimal compatibility ratio of PTX and BEZ235, we designed a Nano-Emulsification of paclitaxel and BEZ235 (NEs-PTX-BEZ235) with high efficacy, low toxicity and high patient compliance.\u003c/p\u003e \u003cp\u003eIn this study, a low energy self-emulsification method was used to prepare a Nano-Emulsification of paclitaxel and BEZ235. This emulsifier has uniform particle size distribution, stable properties, and can be fully absorbed by tumor cells. In vitro anti-tumor experiments show that this emulsifier has good effects of synergistically inhibiting tumor proliferation, promoting apoptosis, reducing tumor drug resistance and reversing tumor cell stemness, and provides a promising anti-tumor strategy for future clinical treatment of colon cancer, especially drug-resistant colon cancer.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eOxaliplatin-resistant (HCT116-LOHP) and cisplatin-resistant (HT29-DDP) human colon cancer cell lines were purchased from Shanghai Aurora Technology Co., LTD. HCT116 and HT29 human colon cancer cell lines were purchased from Shanghai Fuxiang Biotechnology Co., LTD. Cisplatin and oxaliplatin, used to maintain drug resistance in two colon cancers, were purchased from Sigma in the United States. BEZ235 and PTX were purchased from Selleck Chemical, USA. Oxaliplatin and cisplatin are dissolved in saline, and PTX and BEZ235 are stored in dimethyl sulfoxide. Sources of other reagents related to cell culture are as follows: DMEM medium (Invitgen, USA), 0.25% EDTA trypsin (Solarbio, Beijing, China), fetal bovine serum (Biolnd, Israel), penicillin-streptomycin mixture (Solarbio, Beijing, China), dimethyl sulfoxide (Solarbio, Beijing, China), PBS (Shengong, Shanghai, China). CCK-8 cell proliferation toxicity assay kit (Tong Ren Chemical Research Institute, Japan) for cell proliferation, Nile Red (Solarbio, Beijing, China), FITC-labeled anti-rabbit IgG (ABCAM, USA), cell cycle assay kit (purchased from Unitech Biotechnology, China). Western blotting detected the antibody, and its concentration was PI3K (1:1000, Abcam, USA), p-Akt (1:2000, Abcam, USA), CD44 (1:2000, Abcam, USA), CD133 (1:1000, Abcam, USA), respectively. USA), Pgp(1:1000, Cst, USA), ABCC1(1:1000, Cst, USA), β-actin (1:1000, Abcam, USA); Hanks Buffer, Type Ⅳ collagenase, Tween 20 and propylene glycol were all purchased from Beijing Solarbio, China, Capryol 90 (Gattefosse, France).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Synthesis of NEs-PTX-BEZ235\u003c/h2\u003e \u003cp\u003ePTX and BEZ235 were dissolved in DMSO, and Tween 20 and propylene glycol were first mixed into the emulsifier by vortex shaking (where the volume ratio of Tween 20 to propylene glycol was 2:1 and shaken for about 3\u0026ndash;5 min); then the double drug (PTX and BEZ235 were added in different concentration ratios of 1:1, 1:2, 1:4, 1:8 and 1:10) was added with Capryol 90 was added to the emulsifier and vortex shaken for 5 min; finally, the above mixture was slowly added to double distilled water and whirlpool shaken for 5 min (volume ratio of Capryol 90, emulsifier and water was 20:40:40) to form the nano complex formulation. The average particle size and stability of the nano complexed formulations (1:1\u0026thinsp;~\u0026thinsp;1:10) were examined by a Malvern laser particle size analyzer, and the prepared nano complexed formulations were stored at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 CCK-8 cell viability determination\u003c/h2\u003e \u003cp\u003eThe four cell lines were cultured in DMEM medium containing 10% fetal bovine serum and 5% penicillin/streptomycin. The HT29DDP cell-resistant line and the HCT116-LOHP cell-resistant line were supplemented with cisplatin (1.5 \u0026micro;g/mL) and oxaliplatin (10 \u0026micro;g/mL), respectively, in the medium to maintain the corresponding resistant phenotypes of the two drug-resistant cells. Cells were inoculated on 96-well plates at a cell density of 4\u0026times;103 cells/well. After 24 hours, the original DMEM medium was removed and replaced with fresh DMEM medium containing different concentrations of BEZ235, PTX, PTX\u0026thinsp;+\u0026thinsp;BEZ235, NE-PTX, NE-PTX combined with BEZ235, NEs-PTX-BEZ235 (1:1, 1:2, 1:4, 1:8 and 1:10 groups) and albumin-paclitaxel (Nab-PTX), respectively. Among them, general cell lines were mixed with PTX or NE-PTX medium of different concentrations using 25 nm BEZ235. In contrast, drug-resistant cell lines were mixed with PTX or NE-PTX medium of different concentrations using 50 nm BEZ235 on 96-well plates. After 48h of treatment, 10\u0026micro;L CCK-8 reagent was added to each well under light protection, and the 96-well plate was wrapped in aluminum foil and placed in the incubator. Two hours later, the absorbance OD value was measured at A450 nm using the BIO-RAD xMark microplate instrument, and the cell survival rate was calculated according to the following formula:\u003c/p\u003e \u003cp\u003eCell survival rate% = (absorbance OD value of experimental group-absorbance of blank group) \u0026divide; (absorbance OD value of control group-absorbance of blank group) \u0026times; 100%\u003c/p\u003e \u003cp\u003eA combination index was calculated to determine the synergetic effect of combination treatment based on the following formula:\u003c/p\u003e \u003cp\u003eCI =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{a}\\text{v}\\text{a}\\text{i}\\text{l}\\text{a}\\text{b}\\text{i}\\text{l}\\text{i}\\text{t}\\text{y}\\:\\text{o}\\text{f}\\:\\text{B}\\text{E}\\text{Z}235}{\\text{C}\\text{e}\\text{l}\\text{l}\\:\\text{a}\\text{v}\\text{a}\\text{i}\\text{l}\\text{a}\\text{b}\\text{i}\\text{t}\\text{y}\\:\\text{o}\\text{f}\\:\\text{N}\\text{E}-\\text{P}\\text{T}\\text{X}\\times\\:\\:\\text{c}\\text{e}\\text{l}\\text{l}\\:\\text{a}\\text{v}\\text{a}\\text{i}\\text{l}\\text{a}\\text{b}\\text{i}\\text{l}\\text{i}\\text{t}\\text{y}\\:\\text{o}\\text{f}\\:\\text{B}\\text{E}\\text{Z}235}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eCI\u0026thinsp;\u0026lt;\u0026thinsp;1, = 1 and \u0026gt;\u0026thinsp;1 indicated synergic, additive and antagonistic effect, respectively\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 In vitro cellular uptake of drugs\u003c/h2\u003e \u003cp\u003eNE-Nile red was prepared by encapsulating Nile red dye in nanoemulsion. Cells were seeded in 6-well plates at a cell density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003ecells/well. After 24 hours, the original medium was removed and replaced by the medium containing NE-Nile red. After 0, 12, 24 and 48 hours of incubation, the plates were washed with PBS, fixed with paraformaldehyde for 15 minutes, washed with PBS again, and incubated with DAPI for 15 minutes to stain the nuclei. Then the uptake of NE-Nile red by tumor cells was observed under a fluorescence microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.5 The expression of multidrug resistance, PI3K/Akt/mTOR pathway related molecules and dry protein were detected by Western blot.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe total protein was extracted after the cells were treated with different drugs for 48 hours. The protein concentration was measured with a nanodrop spectrophotometer. Proteins were separated by Bio-RAD gel electrophoresis and transferred to PVDF membrane. After blocking for 2 H at room temperature (5% bovine serum albumin), the cells were incubated with rabbit anti-human Pgp, ABCC1, PI3K, p-AKt, CD133, CD44 and mouse anti-human β-actin antibodies overnight at 4 ℃, washed at room temperature, and treated with horseradish peroxidase-conjugated anti-rabbit and mouse antibodies for 2 H, then observed and imaged by ECL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Flow cytometry for cell cycle detection experiments\u003c/h2\u003e \u003cp\u003e2 * 10\u003csup\u003e5\u003c/sup\u003e-1 * 10\u003csup\u003e6\u003c/sup\u003ecells were collected, washed once with PBS, and centrifuged to discard the supernatant. Precooled 80% ethanol at -20.degree. C. was added and fixed overnight. On the day of detection, the fixed cells were centrifuged, the supernatant was discarded, washed with PBS, added with 1 ml DNA Staining solution, vortexed for 5\u0026ndash;10 seconds, and incubated for 30 minutes at room temperature in the dark. Detection was performed on a flow cytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Colon cancer primary cell extraction\u003c/h2\u003e \u003cp\u003ePrimary cells were cultured by tissue block attachment method and collagenase digestion method. The fresh tissues of human colorectal cancer were collected and stored in the preservation solution (45ml DMEM\u0026thinsp;+\u0026thinsp;5ml penicillin-streptomycin mixture). The tissues were taken out from the ultra-clean table, fat and necrotic tissues were removed, and washed with Hank's solution. The washed tissues were cut into small pieces of 1mm\u003csup\u003e3\u003c/sup\u003e, washed with Hank's solution again, and the supernatant was discarded. It was resuspended with preservation solution and evenly arranged at the bottom of the culture flask. After adding DMEM culture medium, the culture flask was placed with the bottom upward for 2\u0026ndash;4 hours. When there was cell growth around the tissue block, it was considered that the culture was successful. Collagenase digestion method: The tissue block was cut into small pieces of 1 mm\u003csup\u003e3\u003c/sup\u003e, washed with Hanks solution, added with type IV collagenase, and placed in a 37 ℃ water bath for 1 H until the tissue block was dispersed, and then the cell suspension was collected and filtered through a 200-mesh sterile cell filter. The liquid that passed through the screen was transferred to a flask that had been previously filled with DMEM medium. Cells were cultured for 2\u0026ndash;3 weeks and then purified by trypsin digestion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed by SPSS.20 statistical analysis software, and the results were presented in the form of mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The t-test was used to compare pairwise samples, and the analysis of variance was used to compare multiple samples. The difference was considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Characterization of NEs-PTX-BEZ235\u003c/h2\u003e\n \u003cp\u003eThe ratios of PTX to BEZ235 in the compound were set at 1:1, 1:2, 1:4, 1:8 and 1:10. The Malvern laser particle size analyzer examined the nanoparticle size (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), and the results showed that the particle size distribution of the nano-emulsion (NE) system was narrow, with an average particle size of 171.1 nm and a polydispersion index (PDI) of 0.142\u0026ndash;0.202. The particle size and PDI were similar in alkaline DMEM medium, neutral PBS and pH5.8 buffer. During the subsequent six days of continuous measurements (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), we found that the distribution and particle size of NEs-PTX-BEZ235 remained in the same range without significant changes, especially in the DMEM medium, indicating good stability of NEs-PTX-BEZ235.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 In vitro cellular uptake of drugs\u003c/h2\u003e\n \u003cp\u003eNE-Nile red was prepared by encapsulating Nile red dye in nanoemulsion. In colon cancer cells HT29, HCT116 and colon cancer drug-resistant cells HT29-DDP, HCT116-LOHP, the uptake of NE-Nile red by tumor cells at four time points of 0, 12, 24 and 48 hours was observed. The results showed that NE-Nile red was gradually absorbed and ingested by colon cancer cells and drug-resistant cells with the increase of time. It was almost completely absorbed into the cytoplasm of tumor cells at 48 H (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating that there was a good uptake of NE by colon cancer cells.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.3 Inhibitory effects on tumor cells of single drug group, double drug combination group, nano single drug group, NE-PTX combined with BEZ235 group, NEs-PTX-BEZ235 group and albumin-paclitaxel (Nab-PTX) group\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eUnder the same drug concentration, the effect of the compound preparation 1:10 group was better than that of the other experimental groups, and its IC50 was 2.096 and 1.276 in HT29 and HCT116 cells, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). 9.836 and 6.803 in the drug-resistant cell lines HT29-DDP and HCT116-LOHP, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). The combination index (CI) of NEs-PTX-BEZ235 (1:10) was calculated, demonstrating the synergistic effect of PTX and BEZ235 in the compound preparation.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.4 NEs-PTX-BEZ235 can significantly reduce the expression of PI3K/Akt/mTOR pathway related proteins p-AKt and PI3K in colon cancer cells and drug-resistant cells.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBEZ235 is an inhibitor of PI3K/Akt/mTOR signaling pathway, and blocking PI3K/AKT signaling pathway can enhance the drug sensitivity of drug-resistant human colon cancer cells [1,9]. In this study, Weston blot was used to detect and compare the effects of different drug groups on the expression of PI3K and p-Akt in colon cancer cells and drug-resistant cells. As a result of detection in colon canc cells HT29, HCT116, HT29-DDP, and HCT116-LOHP (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-B), The expressions of PI3K and p-Akt in the BEZ235 single drug group, PTX single drug group, PTX\u0026thinsp;+\u0026thinsp;BEZ235 group, NE-PTX group, NE-PTX combined with BEZ235 group and the NEs-PTX-BEZ235 groups with different double drug ratios are all down-regulated compared with the negative control group, and the down-regulation effect of the NEs-1: 10 group is the most obvious. The difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the inhibitory effect of NEs-1: 10 group was better than that of other experimental groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 NEs-PTX-BEZ235 preparation can significantly reduce the expression of CD44 and CD133 in colon cancer cells.\u003c/h2\u003e\n \u003cp\u003eBEZ235 can inhibit the growth of colon cancer stem cells [1,10]. In order to further explore the possible mechanism of tumor inhibition by drugs, Weston blot was used to detect and compare the effects of different drug groups on the expression of stemness related proteins CD133 and CD44 in colon cancer cells and drug-resistant cells. As a result, in the colon cancer cells HT29, HCT116, HT29-DDP, and HCT116-LOHP (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). Compared with the negative control group, the expressions of CD133 and CD44 in the BEZ235 single drug group, PTX single drug group, PTX combined with BEZ235 group, NE-PTX group, NE-PTX combined with BEZ235 group and NEs-PTX-BEZ235 groups with different double drug ratios were all down-regulated, and the down-regulation effect of the NEs-1: 10 group was the most obvious. The difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the inhibitory effect of NEs-1: 10 group was better than that of other experimental groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 NEs-PTX-BEZ235 can significantly reduce the expression of Pgp and ABCC1 protein in drug-resistant cells.\u003c/h2\u003e\n \u003cp\u003eChemotherapy for colon cancer often leads to the mutation of some genes in the tumor, or stimulates normal cells to produce some chemical factors suitable for tumor growth, thus causing multidrug resistance [11]. In order to explore the mechanism of PTX and BEZ235 on drug-resistant colon cancer cells, we compared the expression of P-glycoprotein (Pgp) and multidrug resistance-associated protein (ABCC1) in different treatment groups by Western blotting. It was found that in HT29DDP and HCT116-LOHP (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE), Compared with the negative control group, the expressions of Pgp and ABCC1 in the BEZ235 single drug group, PTX single drug group, PTX combined with BEZ235 group, NE-PTX group, NE-PTX combined with BEZ235 group and NEs-PTX-BEZ235 groups with different double drug ratios are all down-regulated, and the down-regulation effect of the NEs-1: 10 group is the most obvious. The difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the inhibitory effect of NEs-1: 10 group was better than that of other experimental groups.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.7 Flow cytometry was used to analyze and compare the effects of different proportions of NEs-PTX-BEZ235 on the cell cycle of colon cancer cells and their drug-resistant cells.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eIn the flow cytometry results of colon cancer cells HT29 and HCT116 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-B) and colon cancer drug-resistant cells HT29-DDP (Figure) and HCT116-LOHP (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC-D), it was found that, compared with the untreated control group, NEs-PTX-BEZ235 groups could induce G2/M phase arrest of cells, thereby promoting apoptosis of tumor cells. Among them, the NEs-1: 10 group had the best apoptosis-promoting effect, and the difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE), and the effect was better at high concentration, which also indicated that the induction of G2/M phase arrest by nano-compound drugs on colon cancer cells and their drug-resistant cells was concentration-dependent.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.8 NEs-PTX-BEZ235 has significant inhibitory effect on primary colon cancer cells\u003c/h2\u003e\n \u003cp\u003eThe primary cells of colorectal cancer were extracted from human colorectal cancer specimens and purified by trypsin digestion after successful culture, and the extracted cells were tested by flow cytometry. The CCK-8 method was used to detect the primary colon cancer cells treated with the gradient concentration of NEs-PTX-BEZ235, and the results showed that in the primary colon cancer cells, after 48 hours of drug administration, under the same drug concentration, the survival rate of tumor cells in the NEs-1:10 group decreased more significantly (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). The IC50 values of PTX/BEZ235 nanocomposites (1:1, 1:2, 1:4, 1:8 and 1:10) were 5. 371 nM, 3.813 nM, 4.475 nM, 3.857 nM and 3. 471 nM, respectively.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePTX is considered to be one of the most successful natural anticancer drugs [12], which can promote the assembly of tubulin into microtubules, prevent the dissociation of microtubules, and induce G2/M arrest of colon cancer cells, thereby blocking cell cycle progression, preventing mitosis, inducing the activation of caspase8 apoptosis pathway, and inhibiting cancer cell growth [4,13]. However, PTX has low solubility in water, many side effects, and no targeting [14], which greatly limits its clinical application. BEZ235 is a dual inhibitor of PI3K/Akt/mTOR signaling pathway, which down-regulates the expression of ATP-binding transporters (such as Pgp), reverses Pgp-mediated multidrug resistance and promotes apoptosis. It has been reported that BEZ235 can enhance the sensitivity of colon cancer cell line HCT-116 to 5-fu and inhibit the growth of colon cancer stem cells [1,9]. Combination therapy is a common strategy to overcome drug resistance. Therefore, we speculate that BEZ235 and PTX may act synergistically on multiple targets of colon cancer cells, thereby reducing drug resistance and further promoting apoptosis. Nanoemulsion is a stable, transparent, low-viscosity, uniform and thermodynamically stable dispersion system composed of oil phase, water phase, surfactant and cosurfactant in a certain proportion, which can increase the solubility of poorly soluble drugs and improve the stability and bioavailability of drugs [22,23]. In our previous studies, it has been proved that the combination of PTX and BEZ235 has a good synergistic inhibitory effect on the growth of colon cancer cells with K-ras mutation +/-and their drug-resistant cells, and the application of nanoemulsion loaded with PTX combined with low concentration of BEZ235 can further enhance the efficacy [8].\u003c/p\u003e \u003cp\u003eThe purpose of this study is to complete the design and development of NEs-PTX-BEZ235 compound preparation according to the optimal proportion, verify its stability, explore its anti-tumor effect and mechanism of action, and verify it in clinical samples, so as to lay a foundation for exploring its potential clinical application value.\u003c/p\u003e \u003cp\u003eFirstly, PTX and BEZ235 were wrapped into nanoemulsion according to different concentration ratios of 1: 1,1: 2,1: 4,1: 8 and 1:10, referring to the previous preparation method of paclitaxel loaded nanoemulsion, and the nanoparticle size of compound preparation was detected by Malvern laser particle size analyzer. The results showed that the particle size of NEs-PTX-BEZ235 was uniformly distributed in alkaline DMEM medium, neutral PBS and acidic buffer (pH 5.8), the particle size range was 100\u0026ndash;200 nM, the average particle size was 171.1 nM, and the polydispersity index (PDI) was 0.142\u0026ndash;0.202 (\u0026lt;\u0026thinsp;0.3). Indicating that the particle size distribution is relatively concentrated. In the subsequent 6 days of continuous measurement, we found that the particle size distribution of NEs-PTX-BEZ235 did not change significantly, especially in DMEM medium, which indicated that NEs-PTX-BEZ235 had good stability. After that, we detected the uptake of empty nanoemulsion (NE) by tumor cells in vitro by fluorescence experiment, and found that HT29, HCT116, HT29-DDP and HCT116-LOHP cells had good uptake of NE.\u003c/p\u003e \u003cp\u003eThen, we compared the effects of single drug group, double drug combination group, nano single drug group, nano paclitaxel combined with BEZ235 group, NEs-PTX-BEZ235 group and albumin-paclitaxel (Nab-PTX) group on the proliferation of colon cancer HT29, HCT116, HT29-DDP and HCT116-LOHP cells through CCK8 experiment. And calculate that survival rate of the tumor cells. The results showed that both PTX and BEZ235 could inhibit the proliferation of colon cancer cells and their drug-resistant cells in a dose-dependent manner, but when BEZ235 was used alone, the concentration was too high, and the inhibitory effect on drug-resistant colon cancer cells was not obvious. The IC50 of HT29, HCT116, HT29-DDP and HCT116-LOHP cells in the nanoemulsion loaded with PTX or BEZ235 was significantly lower than that in the single drug group, and the difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that after the nanoemulsion was loaded with PTX or BEZ235, The dosage can be reduced; Colon cancer cell lines HT29 and HCT116 and their drug-resistant cell lines HT29-DDP and HCT116-LOHP were treated with gradient concentrations of PTX or NE-PTX combined with low concentrations of BEZ235 in dual-drug combination therapy. The results showed that with the increase of PTX or NE-PTX concentration, The IC50 of tumor cells decreased more significantly than that of single drug group, suggesting that the combination of NE-PTX and BEZ235 can complement each other and enhance each other's sensitivity. When NE-PTX combined with low concentration of BEZ235, it showed more intense tumor inhibition. In that proces of treating colon canc cell strains HT29 and HCT116 and drug-resistant cell strains HT29-DDP and HCT116-LOHP thereof, the nano paclitaxel-BEZ235 compound preparation with gradient concentration and different double-drug matching is apply, When the concentration ratio of NE-PTX and BEZ235 was 1:10, the effect was the best. The IC50 of PTX and BEZ235 in normal colon cancer cell lines was reduced to about 1/12 of that of paclitaxel alone, and the dosage in drug-resistant cell lines was also reduced to about 2/5 of that of paclitaxel alone. The effect was better than that of NE-PTX combined with BEZ235. In addition, the IC50 of the NEs-PTX-BEZ235 was significantly lower than that of albumin-paclitaxel (Nab-PTX), which is currently used in clinical practice.\u003c/p\u003e \u003cp\u003ePI3K/Akt/mTOR signaling pathway is closely related to multidrug resistance of tumors, and plays an important role in the occurrence and development of tumors, which is considered as a new target for chemotherapy resistance treatment [15,16]. The combination of PTX and BEZ235 could effectively inhibit the expression of PI3K/Akt/mTOR pathway-related proteins PI3K and p-Akt, and the inhibitory effect was more obvious when it was prepared into NEs-PTX-BEZ235, and the effect of NES-1: 10 group was the best, with statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There are some special tumor cell subsets in the tumor cell population, which are called cancer stem cells (CSCs) or tumor initiating cells (TICs) [17,18]. These cells have malignant biological potentials such as self-renewal, multi-directional differentiation, unlimited proliferation and tumor reconstruction, and are considered to be the root of tumorigenesis, progression, invasion and metastasis, chemoradiotherapy resistance and recurrence [19,20]. CD44 and CD133 can be used as markers of colon cancer stem cells. Through Western blotting experiments, we found that compared with other experimental groups, The inhibition of CD44 and CD133 expression in colon cancer cells HT29, HCT116 and their drug-resistant cells HT29-DDP, HCT116-LOHP by NEs-1:10 group was the best, and the difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). ABCC1/MRP1 and ABCB1/Pgp are energy-dependent drug efflux pumps with an ATP-binding site and a drug-binding site. ATP can be hydrolyzed by binding to ABCC1 and Pgp to provide energy. This energy is used to pump out the drug entering the cell, resulting in the failure of the intracellular drug concentration to reach an effective level, thus causing drug resistance and leading to chemotherapy failure [8,21]. Our previous study demonstrated that the combination of PTX and BEZ235 can effectively inhibit the expression of drug resistance proteins ABCC1 and Pgp in colon cancer cells [8]. In this study, we also proved that the NEs-PTX-BEZ235 could down-regulate the expression of ABCC1 and Pgp in drug-resistant colon cancer cell lines, and the down-regulation effect of NEs-1: 10 group was the most obvious, and the difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PTX can induce G2/M phase arrest and activation of caspase8 apoptosis pathway in colon cancer cells [13]. NEs-PTX-BEZ235 can also induce G2/M arrest of colon cancer cells HT29, HCT116 and colon cancer drug-resistant cells HT29-DDP, HCT116-LOHP, thus inhibiting the growth of tumor cells, and the NEs-1: 10 group has the best effect.\u003c/p\u003e \u003cp\u003eTo sum up, our experimental results show that NEs-PTX-BEZ235 can effectively improve the drug sensitivity of colon cancer cells and drug-resistant cells, and its synergistic killing effect may be related to promoting the programmed death process of tumor cells and inhibiting the stemness and drug resistance of colon cancer cells. It was found that this emulsifier had the same obvious anti-cancer effect in human colon cancer primary cells, suggesting that the compound preparation is expected to become an effective means for the treatment of drug-resistant colon cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by the National Natural Science Foundation of China (grant numbers 82373176) and the Zhejiang Province Basic Public Welfare Research Program Project (grant number LY23H160012). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXingyao Zhu: Data curation, Formal analysis, Methodology, Validation, Writing - original draft. Ruiqi Zhang: Methodology, Resources, Writing - original draft. Yuhua Liu: Data curation. Caiyun Yang: Data curation. Qi Ding: Data curation. Wenyi Gu: Supervision. Hong Zou: Conceptualization, Formal analysis, Funding acquisition, Project administration.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRoper J, Richardson MP, Wang WV, et al. The dual PI3K/mTOR inhibitor NVP-BEZ235 induces tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer. PloS one. 2011;6:e25132.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLidi Z, Zhen L, Bin J, et al. Research progress of NVP-BEZ235 combined with anti-tumor drugs. Basic Clin Med. 2016;36:1450-4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGang C, Qian Z, Min W, et al. Research progress of endophytic fungi producing taxol. Chinese Wild Plant Resources. 2015;34:24\u0026thinsp;\u0026minus;\u0026thinsp;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePham DT, Saelim N, Tiyaboonchai W. Paclitaxel loaded EDC-crosslinked fibroin nanoparticles: a potential approach for colon cancer treatment. 2020;10:413\u0026thinsp;\u0026minus;\u0026thinsp;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhiwen L, Zhangjun Q. Progress in clinical application of paclitaxel liposomes. Asia-Pac Trad Med. 2012;8:229\u0026thinsp;\u0026minus;\u0026thinsp;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaitao Y. Progress in synthesis and application of anticancer drug paclitaxel. Heilongjiang Science and Technology Information. 2017:129.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou H, Li L, Garcia Carcedo I, et al. Synergistic inhibition of colon cancer cell growth with nanoemulsion-loaded paclitaxel and PI3K/mTOR dual inhibitor BEZ235 through apoptosis. International journal of nanomedicine. 2016;11:1947-58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu Y, Zhang K, Zhu X, et al. Synergistic Inhibition of Drug-Resistant Colon Cancer Growth with PI3K/mTOR Dual Inhibitor BEZ235 and Nano-Emulsioned Paclitaxel via Reducing Multidrug Resistance and Promoting Apoptosis. 2021;16:2173-86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen YH, Wang CW, Wei MF, et al. Maintenance BEZ235 Treatment Prolongs the Therapeutic Effect of the Combination of BEZ235 and Radiotherapy for Colorectal Cancer. 2019;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J, Shao R, Li F, et al. PI3K/Akt/mTOR pathway dual inhibitor BEZ235 suppresses the stemness of colon cancer stem cells. Clinical and experimental pharmacology \u0026amp; physiology. 2015;42:1317-26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang YK, Wang YJ, Lei ZN, et al. Regorafenib antagonizes BCRP-mediated multidrug resistance in colon cancer. Cancer letters. 2019;442:104\u0026thinsp;\u0026minus;\u0026thinsp;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeaver BA. How Taxol/paclitaxel kills cancer cells. Molecular biology of the cell. 2014;25:2677-81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTiwari S, Tirosh B, Rubinstein A. Increasing the affinity of cationized polyacrylamide-paclitaxel nanoparticles towards colon cancer cells by a surface recognition peptide. International journal of pharmaceutics. 2017;531:281\u0026thinsp;\u0026minus;\u0026thinsp;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGon\u0026ccedil;alves A, Braguer D, Carles G, et al. Caspase-8 activation independent of CD95/CD95-L interaction during paclitaxel-induced apoptosis in human colon cancer cells (HT29-D4). Biochemical pharmacology. 2000;60:1579-84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArnold M, Sierra MS, Laversanne M, et al. Global patterns and trends in colorectal cancer incidence and mortality. Gut. 2017;66:683\u0026thinsp;\u0026minus;\u0026thinsp;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsuno J, Kanamaru T, Arai K, et al. Synthesis and characterization of nanoemulsion-mediated core crosslinked nanoparticles, and in vivo pharmacokinetics depending on the structural characteristics. Journal of controlled release : official journal of the Controlled Release Society. 2020;324:405\u0026thinsp;\u0026minus;\u0026thinsp;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDreesen O, Brivanlou AH. Signaling pathways in cancer and embryonic stem cells. Stem cell reviews. 2007;3:7\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHennessy BT, Smith DL, Ram PT, et al. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nature reviews Drug discovery. 2005;4:988\u0026ndash;1004.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeltbower RG, Picton S, Bridges LR, et al. Epidemiology of central nervous system tumors in children and young adults (0\u0026ndash;29 years), Yorkshire, United Kingdom. Pediatric hematology and oncology. 2004;21:647\u0026thinsp;\u0026minus;\u0026thinsp;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiang H, Jun D, Yude Z, et al. Isolation and culture of tumor stem cells from human glioma tissue. Chinese Journal of Oncology. 2006:331-3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBao S, Wu Q, McLendon RE, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006;444:756\u0026thinsp;\u0026minus;\u0026thinsp;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEramo A, Ricci-Vitiani L, Zeuner A, et al. Chemotherapy resistance of glioblastoma stem cells. Cell death and differentiation. 2006;13:1238-41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Sun X, Feng Y, et al. Dihydromyricetin reverses MRP2-induced multidrug resistance by preventing NF-κB-Nrf2 signaling in colorectal cancer cell. Phytomedicine : international journal of phytotherapy and phytopharmacology. 2021;82:153414.\u003c/span\u003e\u003c/li\u003e\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"colon cancer, Paclitaxel, BEZ235, Nano-Emulsification, Anti-tumor drug resistance","lastPublishedDoi":"10.21203/rs.3.rs-5424111/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5424111/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs the most common malignant tumor, colorectal cancer is a serious threat to people's health, especially the problem of drug resistance, is a huge challenge. In previous studies, we have found synergistic anti-colon cancer effects with the combination of paclitaxel and BEZ235, especially for drug-resistant colorectal cancer with excellent anti-tumor effects. Therefore, this study aims to investigate the preparation of Nano-Emulsification of paclitaxel and BEZ235 (NEs-PTX-BEZ235), and to explore the potential key parameters of its clinical transformation. To provide a new therapeutic strategy for colorectal cancer, especially for drug-resistant colorectal cancer. The nanomaterials were prepared by low energy self-emulsification method, and analyzed by Malvern laser particle size analyzer. Cell drug uptake was observed under fluorescence microscope. CCK8, Western blot and flow cytometry were used to compare the anti-cancer effects and mechanisms of different experimental groups on ordinary and drug-resistant colon cancer cells. Human colon cancer primary cells were extracted to verify the anti-tumor effect of drugs.\u003c/p\u003e \u003cp\u003eFinally, we found that NEs-PTX-BEZ235 significantly killed colon cancer cells, especially drug-resistant cells, and better than Nab-PTX. It may play a synergistic role in reducing drug resistance, inhibiting tumor stemness and inducing apoptosis of colon cancer cells by inhibiting drug resistance proteins.\u003c/p\u003e","manuscriptTitle":"Nano-Emulsification of PTX and BEZ235 inhibit colon cancer growth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-12 17:54:19","doi":"10.21203/rs.3.rs-5424111/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-09T06:34:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-07T12:53:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338578794489192648812604890679937996915","date":"2024-12-04T19:45:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-30T23:56:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"335875561934418905700942530741766303599","date":"2024-11-30T20:20:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-30T12:47:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-30T12:24:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-14T05:30:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-13T09:15:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-11-10T03:29:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fdc1ed96-6307-4e53-85cb-ee812687015c","owner":[],"postedDate":"December 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41323722,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":41323723,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2025-05-12T16:01:40+00:00","versionOfRecord":{"articleIdentity":"rs-5424111","link":"https://doi.org/10.1038/s41598-025-94620-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-05-09 15:57:28","publishedOnDateReadable":"May 9th, 2025"},"versionCreatedAt":"2024-12-12 17:54:19","video":"","vorDoi":"10.1038/s41598-025-94620-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-94620-y","workflowStages":[]},"version":"v1","identity":"rs-5424111","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5424111","identity":"rs-5424111","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}