Development of Clay-Based Multifunctional Nanomedicine Loaded with the C-Terminal Domain Trun2 of rhCNB for Targeted Colon Cancer Treatment

Development of Clay-Based Multifunctional Nanomedicine Loaded with the C-Terminal Domain Trun2 of rhCNB for Targeted Colon Cancer Treatment | 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 Development of Clay-Based Multifunctional Nanomedicine Loaded with the C-Terminal Domain Trun2 of rhCNB for Targeted Colon Cancer Treatment Hongcui Ma, Li Tong, Ziwei Zhu, Huinan Yang, Jinju Yang, Qun Wei This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8102547/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Recombinant human calcineurin B subunit (rhCNB) has emerged as a promising antitumour therapeutic candidate. Extensive research has shown that the C-terminal region (residues 85–169) of rhCNB, termed Trun2 in this study, serves as the key functional domain responsible for both its antitumour activity and tumour-targeting ability. Targeted drug delivery is a promising strategy for enhancing treatment efficacy while reducing the systemic toxicity and multidrug resistance associated with conventional chemotherapy. In this article, we developed multifunctional layered double hydroxide (LDH) nanoparticles coloaded with Trun2 and paclitaxel (PTX) or doxorubicin (DOX) for the targeted treatment of colon cancer. LDH-PTX-Trun2 or LDH-DOX-Trun2 nanoparticles effectively suppressed colon cancer cell proliferation both in vitro and in vivo. Furthermore, these nanoparticles promoted the secretion and production of antitumour cytokines by bone marrow-derived dendritic cells (BMDCs) and improved tumour site-specific drug delivery. These findings indicated that Trun2 is a promising tumour-targeting small molecular protein for nanoparticle-based drug delivery systems, offering a strategic approach to improving therapeutic precision and efficacy. rhCNB Trun2 LDH nanoparticles targeted delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Calcineurin (CN) is a calcium- and calmodulin-dependent serine/threonine protein phosphatase that consists of catalytic subunit A and regulatory subunit B. The primary role of the calcineurin B subunit (CNB) is to regulate the activity and function of catalytic subunit A [ 1 – 2 ]. Recombinant human CNB (rhCNB) has recently emerged as a promising antitumour therapeutic candidate and is currently in phase I-II clinical trials [ 3 ]. The antitumour activity of rhCNB is associated with its ability to activate and promote the maturation of dendritic cells and macrophages in vivo [ 4 – 5 ]. Furthermore, studies have shown that exogenous rhCNB can enter multiple cell types in vitro and has specific tumour-targeting effects in vivo [ 6 ]. CNB in mammalian cells is composed of 169 amino acids and has 4 EF-hand structures and belongs to the calcium binding protein family. The EF-hand typically has a spiral structure, and each EF-hand structure can bind to a calcium ion. Different EF-hand regions have different binding affinities for calcium ions [ 7 ]. Previous studies have demonstrated that the C-terminal region (residues 85–169), termed Trun2 in this work, is the key domain responsible for the cellular internalization and tumour-targeting properties of rhCNB [ 9 ]. Compared with bulkier targeting ligands such as antibodies, the Trun2 protein offers considerable advantages for drug delivery, including a smaller size, lower production cost, rapid cellular uptake, and superior tumour penetration, thereby enabling more precise and effective therapeutic interventions [ 9 ]. PTX and DOX, which are conventional chemotherapeutic agents, are very strong anticancer drugs, but their indiscriminate cytotoxicity in healthy tissues has restricted their application [ 10 – 13 ]. Furthermore, cancer cells utilize their own transport proteins to pump chemotherapeutic agents out of the cell, leading to multidrug resistance (MDR). Multidrug resistance is among the major obstacles associated with the use of chemotherapy drugs. After long-term chemotherapy, many patients develop multidrug resistance, resulting in reduced drug efficacy or even treatment failure [ 14 ]. Therefore, delivering effective chemotherapeutic agents directly to the tumour site is an efficient strategy to maximize the therapeutic potential of these drugs [ 15 ]. The application of nanoparticles modified with targeting ligands to deliver chemotherapeutic agents directly to tumour sites has attracted considerable interest. These nanoparticles are solid colloidal particles, typically approximately 100 nm in size, composed of natural or synthetic polymers. They facilitate targeted delivery of diverse therapeutic molecules, such as hydrophilic and hydrophobic small-molecule drugs, vaccines, and biomacromolecular agents. Various types of nanocarriers offer substantial benefits, including enhanced efficacy, improved safety profiles, optimized physicochemical properties, and reduced systemic side effects of chemotherapeutic treatments [ 16 – 17 ]. Layered double hydroxides (LDHs) have emerged as promising platforms for drug delivery. LDHs are a class of anionic clays that exhibit exceptional chemical stability, excellent biocompatibility, tunable drug-loading capacity, and pH-responsive release properties, making them particularly suitable for targeted cancer therapy [ 18 – 20 ]. In this study, we constructed a multifunctional LDH-based nanomedicine by loading Trun2 and either PTX or DOX onto the same LDH NPs for colon cancer therapy. The targeting efficiency, immunomodulation effects, and antitumour efficacy were evaluated in vitro and in vivo experiments. This research is expected to provide a promising strategy for the design of advanced targeted delivery systems for cancer treatment. 2. Materials and methods 2.1 Materials and reagents MgCl2·6H2O, AlCl3·9H2O, and NaOH were purchased from Beijing Chemicals (Beijing, China). Basic RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS), penicillin − streptomycin, and trypsin were purchased from Corning (USA). 5-Diphenyltetrazolium bromide (MTT) and DAPI were purchased from Solarbio Life Sciences (China). PTX, DOX, 5-FAM and Cy7 were purchased from Nanjing Goyoo Biotech Co., Ltd. Bovine serum albumin (BSA) was purchased from Sigma‒Aldrich (USA). 2.2 Cell lines and animals The mouse colon carcinoma cell line CT26 and the human colon carcinoma cell line RKO were purchased from the American Type Culture Collection (ATCC, USA). All the cell lines were confirmed to be free of Mycoplasma contamination by PCR. Female ICR mice (6–8 weeks old, weighing 30–35 g) were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China). 2.3 Methods 2.3.1 Preparation of LDH-PTX/DOX-Trun2 NPs LDH NPs were synthesized as previously described. Briefly, 10 mL of a mixed solution of MgCl2 (0.7 M) and AlCl3 (0.3 M) was rapidly added to 40 mL of NaOH (0.45 M) solution and stirred vigorously under a nitrogen flow for 30 minutes. The slurry was collected, divided equally into three centrifuge tubes, and centrifuged to obtain the MgAl-Cl-LDH nanoparticles. One pellet was resuspended in an aqueous solution containing either PTX or DOX and incubated in a 37°C shaker for 1 hour to facilitate drug loading. Another pellet was resuspended in ultrapure water. All the pellets were subsequently subjected to hydrothermal treatment at 100°C for 16 hours, yielding n-LDH-PTX, n-LDH-DOX or plain n-LDH NPs. Suspensions of n-LDH, n-LDH-PTX, or n-LDH-DOX nanoparticles were incubated either with BSA (10 mg/mL) alone or with a 4:1 mixture of BSA (10 mg/mL) and Trun2 (10 mg/mL). The corresponding LDH, LDH-PTX, LDH-DOX, LDH-Trun2, LDH-PTX-Trun2, and LDH-DOX-Trun2 NPs were obtained. The morphology of the synthesized LDH NPs was characterized using a Hitachi HT7700 transmission electron microscope (TEM). The hydrodynamic diameter and zeta potential were determined by dynamic light scattering (DLS) on a Malvern Instruments system. 2.3.2 Cellular uptake of LDH-PTX/DOX-Trun2 NPs For the cellular uptake of the nanoparticles, various LDH NP formulations were labelled with 5-FAM. 5-FAM-SE specifically reacts with amino groups to label BSA or BSA-Trun2. Briefly, an equimolar amount of 5-FAM-SE was mixed with BSA or BSA-Trun2 and incubated in the dark at room temperature for 1 h. The resulting mixture was extensively dialyzed overnight at 4°C against PBS using a 10 kDa cut-off dialysis membrane to remove unbound dye. CT26 cells were seeded in 6-mm confocal dishes (4×10⁵ cells/well) and incubated overnight. The cells were then treated with 50 µg/mL 5-FAM-labelled LDH-Trun2, LDH-PTX-Trun2, LDH-DOX-Trun2, or unlabelled LDH-Trun2 nanoparticles (NPs) for 1 h at 37°C. Following incubation, the cells were washed three times with PBS, subjected to acid-stripping buffer (pH 5.0 Gly-HCl buffer), and fixed with 4% paraformaldehyde. Cellular uptake of these nanoparticles was visualized using a Zeiss confocal fluorescence microscope. All experiments were performed in triplicate and independently repeated three times. 2.3.3 Cell cytotoxicity assay CT26 or RKO cells were seeded in 96-well plates at a density of 4×10³ cells/well and allowed to adhere overnight. The following day, the culture medium was replaced with fresh medium containing serial dilutions (0, 12.5, 25, 50, 100, and 200 µg/mL) of the following nanoparticles: LDH, LDH-PTX, LDH-DOX, LDH-Trun2, LDH-PTX-Trun2, or LDH-DOX-Trun2. After 48 hours of exposure, cell viability was assessed using a CCK-8 assay. All treatments were performed in three replicate wells, and the entire experiment was independently repeated three times. 2.3.4 Isolation and immunoactivation of mouse bone marrow-derived DCs The bone marrow cells from C57BL/6 wild-type mice were flushed with RPMI 1640 medium, dispersed and passed through a 200 nylon mesh and centrifuged for 5 min at 1500 × g, after which the erythrocytes were lysed with ACK Lysis Buffer. The remaining cells were washed twice with medium and cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin in the presence of 25 ng/mL recombinant mGM-CSF and 25 ng/mL mIL-4. The medium was changed every 2 days, and a portion of the cells was stained with CD11C every 3 days. On Day 6 of culture, nonadherent and loosely adherent cells were harvested and seeded in 12-well plates; the next day, 200 µg/mL LDH NPs or LDH-Trun2 NPs were added, and the cells were incubated further for 48 hours. The culture supernatants were collected, and cytokine and chemokine levels were determined by ELISA. Three replicates were tested, and each experiment was repeated 3 times. 2.3.5 Distribution of LDH NPs in CT-26 tumour-bearing mice To evaluate the distribution of the nanoparticles in vivo, Cy7-labelled LDH, LDH-Trun2, and LDH-PTX-Trun2 NPs were prepared as described in Section 2.3.2. CT26 tumour-bearing models were established by subcutaneously inoculating 1×10⁶ CT26 cells into the right armpit of BALB/c mice. When the tumour volume reached approximately 100 mm³, the mice were randomly divided into three groups (n = 5 per group for the experimental groups; n = 2 for the control group). The mice were administered 200 µg of the respective Cy7-labelled NPs via tail vein injection. In vivo fluorescence imaging was performed using an IVIS system at 10, 24 and 36 h postinjection. At each time point, one mouse per group was euthanized, and its tumours and major organs (heart, liver, spleen, lungs, and kidneys) were harvested for ex vivo imaging. The fluorescence intensity at the tumour sites was quantified using ImageJ software by measuring the mean grey value. 2.3.6 Tumour treatment A colon tumour model was established by subcutaneously injecting 1×10 6 CT26 cells into female ICR mice (30–35 g, 6–8 weeks old). After tumour implantation, the ICR mice were maintained under standard housing conditions. The tumour volume was measured every 2 days, and once the tumour tissue volume reached ~ 50 mm 3 , the mice were randomly allocated to seven groups (10 mice/group) and intravenously injected with 200 µg of (1) saline, (2) LDH NPs, (3) LDH-PTX NPs, (4) LDH-Trun2 NPs, (5) LDH-PTX-Trun2 NPs, (6) LDH-DOX NPs, or (7) LDH-DOX-Trun2 NPs every 2 days. The tumour volume was recorded every 2 days. After 9 days, the mice were sacrificed, and the tumours were removed and weighed. 2.3.7 Statistical analysis Statistical analysis was performed using one-way analysis of variance (ANOVA) with post hoc Tukey's test for multiple comparisons in GraphPad Prism 6. A p value of < 0.05 was considered to indicate statistical significance; significance levels are denoted as *p < 0.05, **p < 0.01, and ***p < 0.001. 3. Results 3.1 Synthesis and characterization of LDH nanoparticles The LDH nanoparticles synthesized in this study are MgAl nanoparticles with a characteristic “sandwich-like” structure, consisting of positively charged cationic layers composed of Mg² and Al³, with interlayer spaces occupied by negatively charged Cl⁻ anions. As shown in Fig. 1 . A , most of the nanoparticles were plate-like particles with a hexagonal shape. The average particle size of the NPs measured by DLS ranged from 118 nm (LDH) to 194 nm (LDH-PTX-Trun2) (Fig. 1 B), and the lateral dimensions ranged from 50 nm to 200 nm, indicating that the NPs were suitable for cellular uptake. 3.2 Cellular uptake of LDH NPs Our previous studies indicated that Trun2 can be rapidly internalized into tumour cells (unpublished data). To investigate the cellular uptake of Trun2-loaded LDH nanoparticles, we incubated CT26 cells with unlabelled LDH-Trun2, 5-FAM-labelled LDH-Trun2, 5-FAM-labelled LDH-PTX-Trun2, and 5-FAM-labelled LDH-DOX-Trun2. As shown in Fig. 2 A, bright green fluorescence originating from 5-FAM rapidly appeared in the cytoplasm within 15 minutes of incubation, indicating efficient internalization of the Trun2-loaded LDH nanoparticles by CT26 cells with no statistically significant differences among the 5-FAM-labeled groups, implying that the presence of chemotherapeutic drugs (PTX or DOX) did not interfere with the cellular uptake process mediated by Trun2 or the LDH nanocarrier ( Fig. 2 B ) . 3.3 LDH-PTX/DOX-Trun2 NPs inhibit tumour cell proliferation To evaluate the therapeutic efficacy of PTX- or DOX-loaded LDH nanoparticles, cytotoxicity assays were performed in CT26 and RKO cells (Fig. 3 ). In RKO cells, neither plain LDH nor LDH-Trun2 nanoparticles exhibited detectable cytotoxicity. In contrast, compared with the unloaded controls, both PTX- and DOX-loaded nanoparticles (LDH-PTX and LDH-DOX), with or without Trun2 modification, significantly suppressed cell viability at higher concentrations. At the highest concentration tested (200 µg/mL), compared with their nontargeted counterparts, the Trun2-functionalized LDH-PTX or LDH-DOX nanoparticles significantly enhanced the drug cytotoxicity (P < 0.05; Fig. 3 A and 3 B). Compared with PTX-loaded nanoparticles, DOX-loaded nanoparticles exhibited better inhibitory effects. In CT26 cells, compared with the vehicle, both plain LDH and LDH-Trun2 nanoparticles demonstrated significant differences in terms of intrinsic cytotoxicity. Compared with treatment with chemotherapeutic drug-free nanoparticles or Trun2-only nanoparticles, treatment with LDH-PTX or LDH-DOX, as well as their Trun2-modified counterparts, led to a significant reduction in cell viability (Fig. 3 C and 3 D). Compared with LDH-PTX, Trun2-loaded LDH-PTX tended towards increased cytotoxicity, which reached statistical significance at 200 µg/mL (P < 0.01; Fig. 3 C). Morever, a marked difference in cytotoxicity was observed between Trun2-LDH-DOX and LDH-DOX at 12.5 µg/mL (P < 0.001), although this effect was not observed at other concentrations (Fig. 3 D). Furthermore, the LDH NP formulations exhibited stronger inhibitory effects on CT26 cells than on RKO cells, and compared with PTX-loaded NPs, DOX-loaded NPs demonstrated greater cytotoxicity on RKO cells. Specifically, at a concentration of 200 µg/mL, the inhibition rate of Trun2-modified LDH-PTX NPs was only 10%–20%, while that of Trun2-LDH-DOX NPs reached 30%–40%. In contrast, the same treatments led to markedly higher inhibition rates in CT26 cells, with Trun2-LDH-PTX reaching 60%–70% and Trun2-LDH-DOX reaching 30%–40% at the equivalent concentration. Collectively, these results indicate that PTX- and DOX-loaded LDH nanoparticles were effective at killing both CT26 and RKO cells and that their cytotoxicity was further enhanced by Trun2 modification, likely as a result of the increased cell penetration of the NPs, which potentiated the action of the chemotherapeutic drugs. 3.3 LDH NPs induce the production of cytokines by BMDCs Previous studies have demonstrated that Trun2 effectively stimulates and activates bone marrow-derived dendritic cells (BMDCs) to secrete TNF-α, IL-12, and IL-12p70. To investigate whether Trun2-modified LDH nanoparticles enhance the immunostimulatory effects of LDH NPs, we isolated and prepared BMDCs from wild-type ICR mice. The BMDCs were incubated overnight with either plain LDH nanoparticles or Trun2-modified LDH nanoparticles. The culture supernatants were subsequently collected to analyse changes in cytokine levels. As shown in Fig. 4 , compared with those in the controls, the levels of TNF-α, IFN-γ, and IL-12p70 significantly increased in the supernatants of BMDCs treated with both LDH and LDH-Trun2 nanoparticles. Notably, compared with the LDH NP group, the LDH-Trun2 group exhibited a more pronounced increase in cytokine secretion, and the difference was significant ( Fig. 4 ) . These results indicate that LDH nanoparticles inherently possess immunostimulatory properties and that loading with Trun2 further amplifies this immunostimulatory activity. 3.4 In vivo imaging of LDH-PTX-Trun2 nanoparticles Previous evidence has shown that exogenous Trun2 exhibits tumour-targeting properties in vivo (unpublished data). To further evaluate the targeting efficiency of Trun2-loaded LDH nanoparticles (NPs) and to determine whether Trun2 enhances the tumour-targeting ability of LDH-based nanocarriers, CT26 tumour-bearing mice were intravenously injected with the following formulations: unlabelled LDH NPs (blank control), Cy7-labelled LDH NPs, Cy7-labelled LDH-PTX NPs, and Cy7-labelled LDH-PTX-Trun2. The fluorescence signals were tracked at multiple time points post-injection. As shown in Fig. 5 , at the 10-hour post-injection, fluorescence accumulation was clearly visible within tumour regions in the groups that received Cy7-labelled LDH NPs, LDH-PTX NPs, and LDH-PTX-Trun2. The fluorescence intensity in these groups continued to increase over time, indicating continuous nanoparticle accumulation at the tumor site. Notably, the Cy7-labeled LDH-PTX-Trun2 group displayed the strongest fluorescence intensity among all groups, reflecting superior tumor-targeting capability compared with unmodified LDH or LDH-PTX nanoparticles (Fig. 5 C). Ex vivo imaging of resected tumours further confirmed that Trun2 markedly increased the retention of LDH NPs in tumour tissue (Fig. 5 B). Together, these results clearly indicate that Trun2 conjugation markedly improves the tumor-targeting and retention capabilities of LDH-based nanocarriers in vivo. 3.5 Trun2-loaded LDH-PTX/DOX nanoparticles inhibit tumour growth in CT26 tumour-bearing mice The antitumor efficacy of the Trun2-modified LDH nanoparticle formulations was evaluated in CT26 tumor-bearing ICR mice. As shown in Fig. 6 A–D, treatment with unmodified LDH nanoparticles did not significantly inhibit tumor growth compared with the model group, indicating good biocompatibility of the carrier material. In contrast, Trun2-functionalized LDH nanoparticles (LDH-Trun2) exhibited moderate but significant tumor growth suppression ( P < 0.01), demonstrating that surface modification with Trun2 enhanced the inherent antitumor activity of the LDH nanoparticles system. For the paclitaxel-loaded formulations (Fig. 6 A, B), both LDH-PTX and LDH-PTX-Trun2 nanoparticles markedly inhibited tumor growth compared with the saline and LDH control groups ( P < 0.001). Importantly, the LDH-PTX-Trun2 group achieved the most pronounced antitumor effect, resulting in a tumor inhibition rate of approximately 60.9% , which was significantly higher than that observed for LDH-PTX alone ( P < 0.001). The tumor weights at the endpoint were also markedly lower in the LDH-PTX-Trun2 group compared with all other groups ( P < 0.0001), confirming its superior therapeutic efficacy. Similarly, in the doxorubicin-loaded groups (Fig. 6 C, D), LDH-DOX and LDH-DOX-Trun2 nanoparticles significantly suppressed tumor growth compared with the saline and LDH controls ( P < 0.01). The Trun2-modified LDH-DOX nanoparticles further enhanced tumor inhibition relative to the unmodified LDH-DOX formulation ( P < 0.05), achieving a tumor inhibition rate of approximately 50% . Collectively, these findings indicate that Trun2 conjugation enhances the antitumor efficacy of LDH-based nanocarriers. Among all tested formulations, LDH-PTX-Trun2 nanoparticles demonstrated the strongest inhibitory effect on CT26 tumor growth, highlighting their potential as an effective targeted nanotherapeutic system for colon cancer treatment. 4. Discussion Recent advances in ligand-modified nanoparticles for targeted delivery of chemotherapeutic agents have attracted considerable attention from the biomedical community because of their ability to enhance intracellular uptake and enable sustained drug release [ 21 ]. Trun2, a truncated form of rhCNB with established tumour-targeting specificity, has emerged as a highly promising targeting ligand. In this study, we developed a novel tumour-targeted delivery system by conjugating Trun2 to layered double hydroxide nanoparticles (LDH NPs). The resulting Trun2-modified LDH-PTX nanoparticles demonstrated superior therapeutic performance, including significant suppression of CT26 tumour growth. Ligand-mediated targeting not only promoted efficient tumour-selective delivery but also reduced systemic toxicity while enhancing chemotherapeutic efficacy. Importantly, this delivery strategy shows strong potential to overcome chemoresistance, representing a notable advancement in precision cancer nanomedicine [ 21 – 22 ]. The antitumour effects of rhCNB are associated with its ability to activate dendritic cells and macrophages in vivo [ 4 – 5 , 23 ]. Trun2 significantly enhanced the immunomodulatory effects of LDH NPs and promoted the secretion of cytokines. Both TNF-α and IFN-γ are critical immunomodulatory factors that regulate apoptosis and exert cytotoxic or inhibitory effects on tumour cells. IL-12p70 is a key cytokine that enhances the cytotoxic activity of T cells and NK cells while stimulating IFN-γ secretion [ 24 – 25 ]. Therefore, we propose a codelivery strategy using Trun2 and LDH to encapsulate chemotherapeutic agents. This approach aims to synergize the antitumour and targeting effects of chemotherapy drugs with the immunostimulatory properties of LDH and Trun2, thereby achieving enhanced antitumour efficacy. In this study, the antitumour efficacy of Trun2-modified LDH-PTX or LDH-DOX was lower than we anticipated. This limited performance may be attributed to two main factors. First, the loading capacity of PTX or DOX in the LDH nanoparticles was relatively low, resulting in suboptimal therapeutic outcomes in CT26 tumour-bearing mice; second, the actual amount of Trun2 loaded onto the LDH-NPs reached only one-fifth or less of the intended dose, which likely compromised its immunostimulatory effects. In subsequent studies, we plan to explore alternative chemotherapeutic agents and increase the amount of Trun2 loaded onto LDH-NPs to enhance the overall therapeutic response. LDH nanoparticles are two-dimensional anionic clays capable of adsorbing substantial amounts of protein on their surfaces, making them an ideal nanoplatform for enhanced cancer immunotherapy [ 18 , 26 ]. Previous studies have shown that LDH nanoparticles can neutralize H⁺ in the tumour immune microenvironment (TIME) and interfere with tumour cell autophagy [ 27 ]. They also act as potent vaccine adjuvants by capturing tumour antigens in situ, thereby eliciting personalized antitumour immune responses [ 26 ]. On the basis of these properties, Trun2-modified LDH nanoparticles represent an integrated platform that combines targeted drug delivery, tumour immunotherapy, and direct chemotherapeutic action. Trun2-modified LDH nanoparticles are multifunctional and capable of precisely delivering chemotherapeutic agents and promoting immune modulation [ 29 ]. Immune modulation is a critical therapeutic strategy for cancer and autoimmune diseases and aims to balance immune activation and suppression within specific pathological contexts [ 30 ]. Multifunctional NPs allow targeted delivery of drugs, cytokines, and immune checkpoint inhibitors (ICIs) while minimizing systemic toxicity and maximizing therapeutic efficacy, positioning them as promising next-generation nanotherapeutics [31]. In summary, this study establishes Trun2 as an effective target protein for the delivery of various chemotherapeutic drugs and highlights a promising strategy for advanced tumour therapy. 5. Conclusions In this study, we developed novel multifunctional layered double hydroxide (LDH) nanoparticles coloaded with Trun2, a functional domain derived from the antitumour therapeutic candidate rhCNB, and the chemotherapeutic agents paclitaxel (PTX) and doxorubicin (DOX). This system synergistically integrates chemotherapy with immunomodulation for targeted treatment of colon cancer. The incorporation of Trun2 significantly enhanced the inhibitory effects on CT-26 colon tumour cell proliferation both in vitro and in vivo, improved tumour-targeting ability, and further promoted the secretion of antitumour cytokines by dendritic cells, suggesting the induction of long-lasting antitumour immunity. Overall, this Trun2-based nanoplatform represents a considerable advancement in targeted drug delivery, providing a robust and translatable strategy to overcome the limitations of conventional chemotherapy. Declarations Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Funding statement This research was funded by the National Natural Science Foundation of China (82172627). Conflict of interest disclosure The authors declare no conflicts of interest. Ethics statement All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals issued by the China Public Health Service. Mice were anesthetized with isoflurane (2–3% for induction and 1–2% for maintenance) in oxygen prior to all surgical or invasive procedures to minimize pain and distress. For euthanasia, animals were deeply anesthetized with isoflurane followed by cervical dislocation, in accordance with the guidelines of the Animal Ethics Committee of Beijing Normal University and the Guide for the Care and Use of Laboratory Animals . The study protocol was reviewed and approved by the Animal Ethics Committee of Beijing Normal University (Approval No. CLS-EAW-2022-025). All mice were housed in a specific pathogen-free (SPF) facility under controlled temperature and a 12-hour light/dark cycle, with ad libitum access to food and water. Every effort was made to minimize animal suffering and to reduce the number of animals used. Consent to Publish All authors have read and approved the final version of the manuscript and consent to its publication. Consent to participate : Not applicable. Author Information Author and Affiliations 1 Gene Engineering and Biotechnology Beijing Key Laboratory, Department of Biochemistry and Molecular Biology, Beijing Normal University，Beijing, 100875, P. R. of China. 2 National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China; Hongcui Ma 1# , Li Tong 1# , Ziwei Zhu 1 , Huinan Yang 2 , Jinju Yang 2 *, Qun Wei 1 * Author Contributions: Study design was carried out by Jinju Yang and Qun Wei, and the experiments was performed by Ziwei Zhu,.The manuscript was written by Jinju Yang and Ziwei Zhu, All authors have read and agreed to the published version of the manuscript. Hongcui Ma and Li Tong contributed equally. Corresponding Author: Correspondence to Jinju Yang or Qun Wei Dual Publication : This manuscript has not been published previously and is not under consideration for publication elsewhere. Authorship : All listed authors have made substantial, direct, and intellectual contributions to the work and approved it for publication. References Hemenway CS, Heitman J. Calcineurin. structure, function, and inhibition. Cell Biochem Biophys. 1999;30:115–51. http://doi:10.1007/BF02737887 . Klee CB, Ren H, Wang X. Regulation of the calmodulin- stimulated protein phosphatase, calcineurin. J Biol Chem. 1998;273(22):3367–13370. http://doi:10.1074/jbc.273.22.13367 . Jin FZ, Lian ML, Wang X, Wei Q. Studies of the anticancer effect of calcineurin B. Immunopharmacol Immunotoxicol. 2005;27:199–210. https://doi.org/10.1081/IPH-20006 7709 . Li J, Guo J, Su Z, Hu M, Liu W, Wei Q. Calcineurin subunit B activates dendritic cells and acts as a cancer vaccine adjuvant. 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13:49:41","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90218,"visible":true,"origin":"","legend":"","description":"","filename":"46c46ff47b094a16bae68e4e010a005c1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8102547/v1/8611027eb7bcaa1d02e7c6d5.xml"},{"id":100792083,"identity":"78bcb3e1-ce7b-401d-9655-e8280f2268d1","added_by":"auto","created_at":"2026-01-21 12:50:23","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100653,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8102547/v1/6274ad7371fd908e6512edb8.html"},{"id":100792068,"identity":"458f5fcd-d048-42db-a922-988cfe9195ff","added_by":"auto","created_at":"2026-01-21 12:50:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3645195,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of synthesized LDH nanoparticles. \u003c/strong\u003e(A) Representative transmission electron microscopy (TEM) images showing the morphology of (i) plain LDH NPs, (ii) LDH-PTX NPs, (iii) LDH-Trun2-PTX NPs, (iv) LDH-DOX NPs, and (v) LDH-Trun2-DOX NPs. (B) Hydrodynamic size distribution profiles of the various nanoparticle formulations, as determined by dynamic light scattering (DLS).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8102547/v1/5351c51e703b8e6f240fdf0f.png"},{"id":100792069,"identity":"062e3de9-a8b0-4b03-acde-82bdda190a27","added_by":"auto","created_at":"2026-01-21 12:50:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2751140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular uptake of fluorescently labelled LDH nanoparticles by CT26 cells. \u003c/strong\u003eCT26 cells were incubated with 50 μg/mL 5-FAM-labelled LDH formulations for 15 minutes. (A) Control group (unlabelled LDH-Trun2 NPs). (B–D) Representative confocal microscopy images demonstrating the rapid cellular internalization of (B) 5-FAM-labelled LDH-Trun2, (C) LDH-DOX-Trun2, and (D) LDH-PTX-Trun2 nanoparticles. The fluorescence intensity was quantified by mean grey value analysis using ImageJ software. The data are presented as the mean ± SEM from three independent experiments (n=3). The scale bar is 20 μm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8102547/v1/4b7285b9ad821c6f7844ef21.png"},{"id":100792066,"identity":"de6e7ad7-b646-4ee6-b2ef-98e1e19ac2fc","added_by":"auto","created_at":"2026-01-21 12:50:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":647634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of the cytotoxicity of LDH nanoparticle formulations in colon cancer cell lines.\u003c/strong\u003e The cytotoxic effects of various LDH NP formulations were assessed in (A, B) the human colon cancer cell line RKO and (C, D) the mouse colon cancer cell line CT-26. Cells were treated with increasing concentrations of the nanoparticles for 48 hours. Cell viability was measured using the CCK-8 assay. The data are presented as the mean ± SEM of three independent experiments (n=3). Statistical significance is indicated as *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, and ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8102547/v1/c4e59581a285da6f05d0cb79.png"},{"id":100797492,"identity":"dbf36b0d-28f2-4c0e-88fc-d2f28dfa4cce","added_by":"auto","created_at":"2026-01-21 13:49:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":745827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTrun2-modified LDH nanoparticles promoted the secretion of antitumour cytokines by BMDCs. \u003c/strong\u003eImmature BMDCs were stimulated with 200 μg/mL LDH NPs and LDH-Trun2 NPs for 48 hours. The concentrations of (A) TNF-α, (B) IFN-γ, and (C) IL-12p70 in the culture supernatant were measured by commercial ELISA kits. The data represent the mean ± SEM of three independent experiments (n=3). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8102547/v1/6b2e83cd8d837382d5bd1f20.png"},{"id":100797118,"identity":"6bd9b410-6504-4c92-a1ce-a1675741d3f9","added_by":"auto","created_at":"2026-01-21 13:47:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1808504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo distribution and tumour accumulation of various Cy7-labelled LDH nanoparticles. \u003c/strong\u003eNear-infrared fluorescence (NIRF) imaging was performed on CT26 tumour-bearing BALB/c mice after intravenous injection of the NPs. (A) Whole-body in vivo images at 10 h, 24 h, and 36 h. (B) Ex vivo images of resected organs and tumours at 10, 24, and 36 hours postinjection. (C) Quantitative fluorescence intensity (mean grey value) at tumour sites over time, analysed from (A) by ImageJ software. The data are shown as the mean ± SEM (n=3); *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8102547/v1/4046b7862daff622f76464f4.png"},{"id":100797643,"identity":"939189ea-743b-4364-a438-7578840c5be5","added_by":"auto","created_at":"2026-01-21 13:50:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":682991,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo antitumour activity of different nanoparticle (NP) formulations against CT26 colon carcinoma in BALB/c mice. CT26 cells were inoculated into BALB/c mice, and the mice received daily intraperitoneal injections of saline or NPs (10 mg/kg). (A, C) The tumours were harvested and weighed on Day 9. (B, D) Tumour growth curves were generated by measuring tumour volume every two days (volume=0.5×length×width\u003csup\u003e2\u003c/sup\u003e). All the data are shown as the mean ± SEM (n=6–8). *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001 versus the saline control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8102547/v1/43f39cc28c126cae9656343a.png"},{"id":104399727,"identity":"0a5a5a8d-dd54-4aed-a68b-f87879dd4067","added_by":"auto","created_at":"2026-03-11 12:07:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10839494,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8102547/v1/e245d19c-7870-436e-9be6-e747a64b635d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of Clay-Based Multifunctional Nanomedicine Loaded with the C-Terminal Domain Trun2 of rhCNB for Targeted Colon Cancer Treatment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCalcineurin (CN) is a calcium- and calmodulin-dependent serine/threonine protein phosphatase that consists of catalytic subunit A and regulatory subunit B. The primary role of the calcineurin B subunit (CNB) is to regulate the activity and function of catalytic subunit A [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Recombinant human CNB (rhCNB) has recently emerged as a promising antitumour therapeutic candidate and is currently in phase I-II clinical trials [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The antitumour activity of rhCNB is associated with its ability to activate and promote the maturation of dendritic cells and macrophages \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, studies have shown that exogenous rhCNB can enter multiple cell types in vitro and has specific tumour-targeting effects \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCNB in mammalian cells is composed of 169 amino acids and has 4 EF-hand structures and belongs to the calcium binding protein family. The EF-hand typically has a spiral structure, and each EF-hand structure can bind to a calcium ion. Different EF-hand regions have different binding affinities for calcium ions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previous studies have demonstrated that the C-terminal region (residues 85\u0026ndash;169), termed Trun2 in this work, is the key domain responsible for the cellular internalization and tumour-targeting properties of rhCNB [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Compared with bulkier targeting ligands such as antibodies, the Trun2 protein offers considerable advantages for drug delivery, including a smaller size, lower production cost, rapid cellular uptake, and superior tumour penetration, thereby enabling more precise and effective therapeutic interventions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePTX and DOX, which are conventional chemotherapeutic agents, are very strong anticancer drugs, but their indiscriminate cytotoxicity in healthy tissues has restricted their application [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, cancer cells utilize their own transport proteins to pump chemotherapeutic agents out of the cell, leading to multidrug resistance (MDR). Multidrug resistance is among the major obstacles associated with the use of chemotherapy drugs. After long-term chemotherapy, many patients develop multidrug resistance, resulting in reduced drug efficacy or even treatment failure [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, delivering effective chemotherapeutic agents directly to the tumour site is an efficient strategy to maximize the therapeutic potential of these drugs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe application of nanoparticles modified with targeting ligands to deliver chemotherapeutic agents directly to tumour sites has attracted considerable interest. These nanoparticles are solid colloidal particles, typically approximately 100 nm in size, composed of natural or synthetic polymers. They facilitate targeted delivery of diverse therapeutic molecules, such as hydrophilic and hydrophobic small-molecule drugs, vaccines, and biomacromolecular agents. Various types of nanocarriers offer substantial benefits, including enhanced efficacy, improved safety profiles, optimized physicochemical properties, and reduced systemic side effects of chemotherapeutic treatments [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLayered double hydroxides (LDHs) have emerged as promising platforms for drug delivery. LDHs are a class of anionic clays that exhibit exceptional chemical stability, excellent biocompatibility, tunable drug-loading capacity, and pH-responsive release properties, making them particularly suitable for targeted cancer therapy [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, we constructed a multifunctional LDH-based nanomedicine by loading Trun2 and either PTX or DOX onto the same LDH NPs for colon cancer therapy. The targeting efficiency, immunomodulation effects, and antitumour efficacy were evaluated in vitro and in vivo experiments. This research is expected to provide a promising strategy for the design of advanced targeted delivery systems for cancer treatment.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and reagents\u003c/h2\u003e \u003cp\u003eMgCl2\u0026middot;6H2O, AlCl3\u0026middot;9H2O, and NaOH were purchased from Beijing Chemicals (Beijing, China). Basic RPMI 1640 medium, Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM), foetal bovine serum (FBS), penicillin\u0026thinsp;\u0026minus;\u0026thinsp;streptomycin, and trypsin were purchased from Corning (USA). 5-Diphenyltetrazolium bromide (MTT) and DAPI were purchased from Solarbio Life Sciences (China). PTX, DOX, 5-FAM and Cy7 were purchased from Nanjing Goyoo Biotech Co., Ltd. Bovine serum albumin (BSA) was purchased from Sigma‒Aldrich (USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell lines and animals\u003c/h2\u003e \u003cp\u003eThe mouse colon carcinoma cell line CT26 and the human colon carcinoma cell line RKO were purchased from the American Type Culture Collection (ATCC, USA). All the cell lines were confirmed to be free of \u003cem\u003eMycoplasma\u003c/em\u003e contamination by PCR.\u003c/p\u003e \u003cp\u003eFemale ICR mice (6\u0026ndash;8 weeks old, weighing 30\u0026ndash;35 g) were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Methods\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Preparation of LDH-PTX/DOX-Trun2 NPs\u003c/h2\u003e \u003cp\u003eLDH NPs were synthesized as previously described. Briefly, 10 mL of a mixed solution of MgCl2 (0.7 M) and AlCl3 (0.3 M) was rapidly added to 40 mL of NaOH (0.45 M) solution and stirred vigorously under a nitrogen flow for 30 minutes. The slurry was collected, divided equally into three centrifuge tubes, and centrifuged to obtain the MgAl-Cl-LDH nanoparticles. One pellet was resuspended in an aqueous solution containing either PTX or DOX and incubated in a 37\u0026deg;C shaker for 1 hour to facilitate drug loading. Another pellet was resuspended in ultrapure water. All the pellets were subsequently subjected to hydrothermal treatment at 100\u0026deg;C for 16 hours, yielding n-LDH-PTX, n-LDH-DOX or plain n-LDH NPs. Suspensions of n-LDH, n-LDH-PTX, or n-LDH-DOX nanoparticles were incubated either with BSA (10 mg/mL) alone or with a 4:1 mixture of BSA (10 mg/mL) and Trun2 (10 mg/mL). The corresponding LDH, LDH-PTX, LDH-DOX, LDH-Trun2, LDH-PTX-Trun2, and LDH-DOX-Trun2 NPs were obtained. The morphology of the synthesized LDH NPs was characterized using a Hitachi HT7700 transmission electron microscope (TEM). The hydrodynamic diameter and zeta potential were determined by dynamic light scattering (DLS) on a Malvern Instruments system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Cellular uptake of LDH-PTX/DOX-Trun2 NPs\u003c/h2\u003e \u003cp\u003eFor the cellular uptake of the nanoparticles, various LDH NP formulations were labelled with 5-FAM. 5-FAM-SE specifically reacts with amino groups to label BSA or BSA-Trun2. Briefly, an equimolar amount of 5-FAM-SE was mixed with BSA or BSA-Trun2 and incubated in the dark at room temperature for 1 h. The resulting mixture was extensively dialyzed overnight at 4\u0026deg;C against PBS using a 10 kDa cut-off dialysis membrane to remove unbound dye. CT26 cells were seeded in 6-mm confocal dishes (4\u0026times;10⁵ cells/well) and incubated overnight. The cells were then treated with 50 \u0026micro;g/mL 5-FAM-labelled LDH-Trun2, LDH-PTX-Trun2, LDH-DOX-Trun2, or unlabelled LDH-Trun2 nanoparticles (NPs) for 1 h at 37\u0026deg;C. Following incubation, the cells were washed three times with PBS, subjected to acid-stripping buffer (pH 5.0 Gly-HCl buffer), and fixed with 4% paraformaldehyde. Cellular uptake of these nanoparticles was visualized using a Zeiss confocal fluorescence microscope. All experiments were performed in triplicate and independently repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Cell cytotoxicity assay\u003c/h2\u003e \u003cp\u003eCT26 or RKO cells were seeded in 96-well plates at a density of 4\u0026times;10\u0026sup3; cells/well and allowed to adhere overnight. The following day, the culture medium was replaced with fresh medium containing serial dilutions (0, 12.5, 25, 50, 100, and 200 \u0026micro;g/mL) of the following nanoparticles: LDH, LDH-PTX, LDH-DOX, LDH-Trun2, LDH-PTX-Trun2, or LDH-DOX-Trun2. After 48 hours of exposure, cell viability was assessed using a CCK-8 assay. All treatments were performed in three replicate wells, and the entire experiment was independently repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4 Isolation and immunoactivation of mouse bone marrow-derived DCs\u003c/h2\u003e \u003cp\u003eThe bone marrow cells from C57BL/6 wild-type mice were flushed with RPMI 1640 medium, dispersed and passed through a 200 nylon mesh and centrifuged for 5 min at 1500 \u0026times; g, after which the erythrocytes were lysed with ACK Lysis Buffer. The remaining cells were washed twice with medium and cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin in the presence of 25 ng/mL recombinant mGM-CSF and 25 ng/mL mIL-4. The medium was changed every 2 days, and a portion of the cells was stained with CD11C every 3 days. On Day 6 of culture, nonadherent and loosely adherent cells were harvested and seeded in 12-well plates; the next day, 200 \u0026micro;g/mL LDH NPs or LDH-Trun2 NPs were added, and the cells were incubated further for 48 hours. The culture supernatants were collected, and cytokine and chemokine levels were determined by ELISA. Three replicates were tested, and each experiment was repeated 3 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5 Distribution of LDH NPs in CT-26 tumour-bearing mice\u003c/h2\u003e \u003cp\u003eTo evaluate the distribution of the nanoparticles in vivo, Cy7-labelled LDH, LDH-Trun2, and LDH-PTX-Trun2 NPs were prepared as described in Section 2.3.2. CT26 tumour-bearing models were established by subcutaneously inoculating 1\u0026times;10⁶ CT26 cells into the right armpit of BALB/c mice. When the tumour volume reached approximately 100 mm\u0026sup3;, the mice were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;5 per group for the experimental groups; n\u0026thinsp;=\u0026thinsp;2 for the control group). The mice were administered 200 \u0026micro;g of the respective Cy7-labelled NPs via tail vein injection. In vivo fluorescence imaging was performed using an IVIS system at 10, 24 and 36 h postinjection. At each time point, one mouse per group was euthanized, and its tumours and major organs (heart, liver, spleen, lungs, and kidneys) were harvested for ex vivo imaging. The fluorescence intensity at the tumour sites was quantified using ImageJ software by measuring the mean grey value.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.6 Tumour treatment\u003c/h2\u003e \u003cp\u003eA colon tumour model was established by subcutaneously injecting 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e CT26 cells into female ICR mice (30\u0026ndash;35 g, 6\u0026ndash;8 weeks old). After tumour implantation, the ICR mice were maintained under standard housing conditions. The tumour volume was measured every 2 days, and once the tumour tissue volume reached\u0026thinsp;~\u0026thinsp;50 mm\u003csup\u003e3\u003c/sup\u003e, the mice were randomly allocated to seven groups (10 mice/group) and intravenously injected with 200 \u0026micro;g of (1) saline, (2) LDH NPs, (3) LDH-PTX NPs, (4) LDH-Trun2 NPs, (5) LDH-PTX-Trun2 NPs, (6) LDH-DOX NPs, or (7) LDH-DOX-Trun2 NPs every 2 days. The tumour volume was recorded every 2 days. After 9 days, the mice were sacrificed, and the tumours were removed and weighed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.3.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using one-way analysis of variance (ANOVA) with post hoc Tukey's test for multiple comparisons in GraphPad Prism 6. A p value of \u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance; significance levels are denoted as *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Synthesis and characterization of LDH nanoparticles\u003c/h2\u003e \u003cp\u003eThe LDH nanoparticles synthesized in this study are MgAl nanoparticles with a characteristic \u0026ldquo;sandwich-like\u0026rdquo; structure, consisting of positively charged cationic layers composed of Mg\u0026sup2; and Al\u0026sup3;, with interlayer spaces occupied by negatively charged Cl⁻ anions. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003cb\u003eA\u003c/b\u003e, most of the nanoparticles were plate-like particles with a hexagonal shape. The average particle size of the NPs measured by DLS ranged from 118 nm (LDH) to 194 nm (LDH-PTX-Trun2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and the lateral dimensions ranged from 50 nm to 200 nm, indicating that the NPs were suitable for cellular uptake.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Cellular uptake of LDH NPs\u003c/h2\u003e \u003cp\u003eOur previous studies indicated that Trun2 can be rapidly internalized into tumour cells (unpublished data). To investigate the cellular uptake of Trun2-loaded LDH nanoparticles, we incubated CT26 cells with unlabelled LDH-Trun2, 5-FAM-labelled LDH-Trun2, 5-FAM-labelled LDH-PTX-Trun2, and 5-FAM-labelled LDH-DOX-Trun2.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, bright green fluorescence originating from 5-FAM rapidly appeared in the cytoplasm within 15 minutes of incubation, indicating efficient internalization of the Trun2-loaded LDH nanoparticles by CT26 cells with no statistically significant differences among the 5-FAM-labeled groups, implying that the presence of chemotherapeutic drugs (PTX or DOX) did not interfere with the cellular uptake process mediated by Trun2 or the LDH nanocarrier \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 LDH-PTX/DOX-Trun2 NPs inhibit tumour cell proliferation\u003c/h2\u003e \u003cp\u003eTo evaluate the therapeutic efficacy of PTX- or DOX-loaded LDH nanoparticles, cytotoxicity assays were performed in CT26 and RKO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn RKO cells, neither plain LDH nor LDH-Trun2 nanoparticles exhibited detectable cytotoxicity. In contrast, compared with the unloaded controls, both PTX- and DOX-loaded nanoparticles (LDH-PTX and LDH-DOX), with or without Trun2 modification, significantly suppressed cell viability at higher concentrations. At the highest concentration tested (200 \u0026micro;g/mL), compared with their nontargeted counterparts, the Trun2-functionalized LDH-PTX or LDH-DOX nanoparticles significantly enhanced the drug cytotoxicity (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Compared with PTX-loaded nanoparticles, DOX-loaded nanoparticles exhibited better inhibitory effects.\u003c/p\u003e \u003cp\u003eIn CT26 cells, compared with the vehicle, both plain LDH and LDH-Trun2 nanoparticles demonstrated significant differences in terms of intrinsic cytotoxicity. Compared with treatment with chemotherapeutic drug-free nanoparticles or Trun2-only nanoparticles, treatment with LDH-PTX or LDH-DOX, as well as their Trun2-modified counterparts, led to a significant reduction in cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Compared with LDH-PTX, Trun2-loaded LDH-PTX tended towards increased cytotoxicity, which reached statistical significance at 200 \u0026micro;g/mL (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Morever, a marked difference in cytotoxicity was observed between Trun2-LDH-DOX and LDH-DOX at 12.5 \u0026micro;g/mL (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), although this effect was not observed at other concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eFurthermore, the LDH NP formulations exhibited stronger inhibitory effects on CT26 cells than on RKO cells, and compared with PTX-loaded NPs, DOX-loaded NPs demonstrated greater cytotoxicity on RKO cells. Specifically, at a concentration of 200 \u0026micro;g/mL, the inhibition rate of Trun2-modified LDH-PTX NPs was only 10%\u0026ndash;20%, while that of Trun2-LDH-DOX NPs reached 30%\u0026ndash;40%.\u003c/p\u003e \u003cp\u003eIn contrast, the same treatments led to markedly higher inhibition rates in CT26 cells, with Trun2-LDH-PTX reaching 60%\u0026ndash;70% and Trun2-LDH-DOX reaching 30%\u0026ndash;40% at the equivalent concentration.\u003c/p\u003e \u003cp\u003eCollectively, these results indicate that PTX- and DOX-loaded LDH nanoparticles were effective at killing both CT26 and RKO cells and that their cytotoxicity was further enhanced by Trun2 modification, likely as a result of the increased cell penetration of the NPs, which potentiated the action of the chemotherapeutic drugs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 LDH NPs induce the production of cytokines by BMDCs\u003c/h2\u003e \u003cp\u003ePrevious studies have demonstrated that Trun2 effectively stimulates and activates bone marrow-derived dendritic cells (BMDCs) to secrete TNF-α, IL-12, and IL-12p70. To investigate whether Trun2-modified LDH nanoparticles enhance the immunostimulatory effects of LDH NPs, we isolated and prepared BMDCs from wild-type ICR mice. The BMDCs were incubated overnight with either plain LDH nanoparticles or Trun2-modified LDH nanoparticles. The culture supernatants were subsequently collected to analyse changes in cytokine levels. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, compared with those in the controls, the levels of TNF-α, IFN-γ, and IL-12p70 significantly increased in the supernatants of BMDCs treated with both LDH and LDH-Trun2 nanoparticles. Notably, compared with the LDH NP group, the LDH-Trun2 group exhibited a more pronounced increase in cytokine secretion, and the difference was significant \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. These results indicate that LDH nanoparticles inherently possess immunostimulatory properties and that loading with Trun2 further amplifies this immunostimulatory activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 In vivo imaging of LDH-PTX-Trun2 nanoparticles\u003c/h2\u003e \u003cp\u003ePrevious evidence has shown that exogenous Trun2 exhibits tumour-targeting properties in vivo (unpublished data). To further evaluate the targeting efficiency of Trun2-loaded LDH nanoparticles (NPs) and to determine whether Trun2 enhances the tumour-targeting ability of LDH-based nanocarriers, CT26 tumour-bearing mice were intravenously injected with the following formulations: unlabelled LDH NPs (blank control), Cy7-labelled LDH NPs, Cy7-labelled LDH-PTX NPs, and Cy7-labelled LDH-PTX-Trun2. The fluorescence signals were tracked at multiple time points post-injection.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, at the 10-hour post-injection, fluorescence accumulation was clearly visible within tumour regions in the groups that received Cy7-labelled LDH NPs, LDH-PTX NPs, and LDH-PTX-Trun2. The fluorescence intensity in these groups continued to increase over time, indicating continuous nanoparticle accumulation at the tumor site. Notably, the Cy7-labeled LDH-PTX-Trun2 group displayed the strongest fluorescence intensity among all groups, reflecting superior tumor-targeting capability compared with unmodified LDH or LDH-PTX nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Ex vivo imaging of resected tumours further confirmed that Trun2 markedly increased the retention of LDH NPs in tumour tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTogether, these results clearly indicate that Trun2 conjugation markedly improves the tumor-targeting and retention capabilities of LDH-based nanocarriers in vivo.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Trun2-loaded LDH-PTX/DOX nanoparticles inhibit tumour growth in CT26 tumour-bearing mice\u003c/h2\u003e \u003cp\u003eThe antitumor efficacy of the Trun2-modified LDH nanoparticle formulations was evaluated in CT26 tumor-bearing ICR mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;D, treatment with unmodified LDH nanoparticles did not significantly inhibit tumor growth compared with the model group, indicating good biocompatibility of the carrier material. In contrast, Trun2-functionalized LDH nanoparticles (LDH-Trun2) exhibited moderate but significant tumor growth suppression (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), demonstrating that surface modification with Trun2 enhanced the inherent antitumor activity of the LDH nanoparticles system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the paclitaxel-loaded formulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B), both LDH-PTX and LDH-PTX-Trun2 nanoparticles markedly inhibited tumor growth compared with the saline and LDH control groups (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Importantly, the LDH-PTX-Trun2 group achieved the most pronounced antitumor effect, resulting in a tumor inhibition rate of approximately \u003cb\u003e60.9%\u003c/b\u003e, which was significantly higher than that observed for LDH-PTX alone (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The tumor weights at the endpoint were also markedly lower in the LDH-PTX-Trun2 group compared with all other groups (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), confirming its superior therapeutic efficacy.\u003c/p\u003e \u003cp\u003eSimilarly, in the doxorubicin-loaded groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D), LDH-DOX and LDH-DOX-Trun2 nanoparticles significantly suppressed tumor growth compared with the saline and LDH controls (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The Trun2-modified LDH-DOX nanoparticles further enhanced tumor inhibition relative to the unmodified LDH-DOX formulation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), achieving a tumor inhibition rate of approximately \u003cb\u003e50%\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eCollectively, these findings indicate that Trun2 conjugation enhances the antitumor efficacy of LDH-based nanocarriers. Among all tested formulations, LDH-PTX-Trun2 nanoparticles demonstrated the strongest inhibitory effect on CT26 tumor growth, highlighting their potential as an effective targeted nanotherapeutic system for colon cancer treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eRecent advances in ligand-modified nanoparticles for targeted delivery of chemotherapeutic agents have attracted considerable attention from the biomedical community because of their ability to enhance intracellular uptake and enable sustained drug release [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Trun2, a truncated form of rhCNB with established tumour-targeting specificity, has emerged as a highly promising targeting ligand. In this study, we developed a novel tumour-targeted delivery system by conjugating Trun2 to layered double hydroxide nanoparticles (LDH NPs). The resulting Trun2-modified LDH-PTX nanoparticles demonstrated superior therapeutic performance, including significant suppression of CT26 tumour growth. Ligand-mediated targeting not only promoted efficient tumour-selective delivery but also reduced systemic toxicity while enhancing chemotherapeutic efficacy. Importantly, this delivery strategy shows strong potential to overcome chemoresistance, representing a notable advancement in precision cancer nanomedicine [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe antitumour effects of rhCNB are associated with its ability to activate dendritic cells and macrophages in vivo [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Trun2 significantly enhanced the immunomodulatory effects of LDH NPs and promoted the secretion of cytokines. Both TNF-α and IFN-γ are critical immunomodulatory factors that regulate apoptosis and exert cytotoxic or inhibitory effects on tumour cells. IL-12p70 is a key cytokine that enhances the cytotoxic activity of T cells and NK cells while stimulating IFN-γ secretion [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, we propose a codelivery strategy using Trun2 and LDH to encapsulate chemotherapeutic agents. This approach aims to synergize the antitumour and targeting effects of chemotherapy drugs with the immunostimulatory properties of LDH and Trun2, thereby achieving enhanced antitumour efficacy.\u003c/p\u003e \u003cp\u003eIn this study, the antitumour efficacy of Trun2-modified LDH-PTX or LDH-DOX was lower than we anticipated. This limited performance may be attributed to two main factors. First, the loading capacity of PTX or DOX in the LDH nanoparticles was relatively low, resulting in suboptimal therapeutic outcomes in CT26 tumour-bearing mice; second, the actual amount of Trun2 loaded onto the LDH-NPs reached only one-fifth or less of the intended dose, which likely compromised its immunostimulatory effects. In subsequent studies, we plan to explore alternative chemotherapeutic agents and increase the amount of Trun2 loaded onto LDH-NPs to enhance the overall therapeutic response.\u003c/p\u003e \u003cp\u003eLDH nanoparticles are two-dimensional anionic clays capable of adsorbing substantial amounts of protein on their surfaces, making them an ideal nanoplatform for enhanced cancer immunotherapy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previous studies have shown that LDH nanoparticles can neutralize H⁺ in the tumour immune microenvironment (TIME) and interfere with tumour cell autophagy [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. They also act as potent vaccine adjuvants by capturing tumour antigens in situ, thereby eliciting personalized antitumour immune responses [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. On the basis of these properties, Trun2-modified LDH nanoparticles represent an integrated platform that combines targeted drug delivery, tumour immunotherapy, and direct chemotherapeutic action.\u003c/p\u003e \u003cp\u003eTrun2-modified LDH nanoparticles are multifunctional and capable of precisely delivering chemotherapeutic agents and promoting immune modulation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Immune modulation is a critical therapeutic strategy for cancer and autoimmune diseases and aims to balance immune activation and suppression within specific pathological contexts [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Multifunctional NPs allow targeted delivery of drugs, cytokines, and immune checkpoint inhibitors (ICIs) while minimizing systemic toxicity and maximizing therapeutic efficacy, positioning them as promising next-generation nanotherapeutics [31].\u003c/p\u003e \u003cp\u003eIn summary, this study establishes Trun2 as an effective target protein for the delivery of various chemotherapeutic drugs and highlights a promising strategy for advanced tumour therapy.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, we developed novel multifunctional layered double hydroxide (LDH) nanoparticles coloaded with Trun2, a functional domain derived from the antitumour therapeutic candidate rhCNB, and the chemotherapeutic agents paclitaxel (PTX) and doxorubicin (DOX). This system synergistically integrates chemotherapy with immunomodulation for targeted treatment of colon cancer. The incorporation of Trun2 significantly enhanced the inhibitory effects on CT-26 colon tumour cell proliferation both in vitro and in vivo, improved tumour-targeting ability, and further promoted the secretion of antitumour cytokines by dendritic cells, suggesting the induction of long-lasting antitumour immunity. Overall, this Trun2-based nanoplatform represents a considerable advancement in targeted drug delivery, providing a robust and translatable strategy to overcome the limitations of conventional chemotherapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (82172627).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest disclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e issued by the China Public Health Service. Mice were anesthetized with isoflurane (2\u0026ndash;3% for induction and 1\u0026ndash;2% for maintenance) in oxygen prior to all surgical or invasive procedures to minimize pain and distress. For euthanasia, animals were deeply anesthetized with isoflurane followed by cervical dislocation, in accordance with the guidelines of the Animal Ethics Committee of Beijing Normal University and the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe study protocol was reviewed and approved by the Animal Ethics Committee of Beijing Normal University (Approval No. CLS-EAW-2022-025). All mice were housed in a specific pathogen-free (SPF) facility under controlled temperature and a 12-hour light/dark cycle, with ad libitum access to food and water. Every effort was made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final version of the manuscript and consent to its publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eGene Engineering and Biotechnology Beijing Key Laboratory, Department of Biochemistry and Molecular Biology, Beijing Normal University，Beijing, 100875, P. R. of China.\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eNational Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHongcui Ma\u003csup\u003e1#\u003c/sup\u003e, Li Tong\u003csup\u003e1#\u003c/sup\u003e, Ziwei Zhu\u003csup\u003e1\u003c/sup\u003e, Huinan Yang\u003csup\u003e2\u003c/sup\u003e, Jinju Yang\u003csup\u003e2\u003c/sup\u003e*, Qun Wei\u003csup\u003e1\u003c/sup\u003e*\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eStudy design was carried out by Jinju Yang and Qun Wei, and the experiments was performed by Ziwei Zhu,.The manuscript was written by Jinju Yang and Ziwei Zhu, All authors have read and agreed to the published version of the manuscript. Hongcui Ma and Li Tong contributed equally.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Jinju Yang or Qun Wei\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual Publication\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThis manuscript has not been published previously and is not under consideration for publication elsewhere.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eAll listed authors have made substantial, direct, and intellectual contributions to the work and approved it for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHemenway CS, Heitman J. Calcineurin. structure, function, and inhibition. 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Nanoscale. 2024;16:17699\u0026ndash;722.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"rhCNB, Trun2, LDH nanoparticles, targeted delivery","lastPublishedDoi":"10.21203/rs.3.rs-8102547/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8102547/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRecombinant human calcineurin B subunit (rhCNB) has emerged as a promising antitumour therapeutic candidate. Extensive research has shown that the C-terminal region (residues 85\u0026ndash;169) of rhCNB, termed Trun2 in this study, serves as the key functional domain responsible for both its antitumour activity and tumour-targeting ability. Targeted drug delivery is a promising strategy for enhancing treatment efficacy while reducing the systemic toxicity and multidrug resistance associated with conventional chemotherapy. In this article, we developed multifunctional layered double hydroxide (LDH) nanoparticles coloaded with Trun2 and paclitaxel (PTX) or doxorubicin (DOX) for the targeted treatment of colon cancer. LDH-PTX-Trun2 or LDH-DOX-Trun2 nanoparticles effectively suppressed colon cancer cell proliferation both in vitro and in vivo. Furthermore, these nanoparticles promoted the secretion and production of antitumour cytokines by bone marrow-derived dendritic cells (BMDCs) and improved tumour site-specific drug delivery. These findings indicated that Trun2 is a promising tumour-targeting small molecular protein for nanoparticle-based drug delivery systems, offering a strategic approach to improving therapeutic precision and efficacy.\u003c/p\u003e","manuscriptTitle":"Development of Clay-Based Multifunctional Nanomedicine Loaded with the C-Terminal Domain Trun2 of rhCNB for Targeted Colon Cancer Treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 12:50:17","doi":"10.21203/rs.3.rs-8102547/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a08059a8-7687-4b55-8dae-93cb731421b4","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T08:11:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 12:50:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8102547","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8102547","identity":"rs-8102547","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}