AI-guided design of a CXCR4-targeted core–shell nanocarrier for co-delivery of berberine/paclitaxel in cancer therapy

AI-guided design of a CXCR4-targeted core–shell nanocarrier for co-delivery of berberine/paclitaxel in cancer therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article AI-guided design of a CXCR4-targeted core–shell nanocarrier for co-delivery of berberine/paclitaxel in cancer therapy Yeonwoo Jang, Amal Babu, Sahil Chahal, Arathy Vasukutty, James J. Moon, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7307122/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Nov, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract An integrated three-step artificial-intelligence (AI) workflow was used to accelerate the design of a CXCR4-targeted, dual-drug nanocarrier for colorectal-cancer therapy. First, the MD-Syn platform screened 38 approved oncology agents across six human colorectal-cancer cell lines (HCT116, HT29, LoVo, RKO, SW620, SW837) and identified paclitaxel (PTX) as the compound with a > 0.9999 probability of synergy with (BBR). Second, a dual peptide-discovery pipeline that combined CABS-dock flexible docking with AlphaFold-multimer/NeuroSNAP-AI scoring yielded a high-affinity 9-mer stromal-cell-derived factor-1 fragment (CXCR4-binding peptide, CXCR4BP; ipTM = 0.95). The peptide was conjugated to DSPE-PEG₂₀₀₀ to form a targeting liposome (CXCR4BPL). Third, FormulationAI predicted that loading PTX into the CXCR4BPL shell and BBR into phosphonate-functionalized mesoporous silica nanoparticles (FMSNs) would maximize encapsulation. A one-pot self-assembly gave FMSN(BBR)-CXCR4BPL(PTX) core–shell nanocarrier (~ 130 nm) with experimental entrapment efficiencies of 78.8 ± 1.9% for BBR and 75.2 ± 2.4% for PTX and sustained co-release over 72 h at pH 7.4. In CXCR4-positive CT26 cells, the core-shell nanocarrier curtailed scratch-wound closure by ~ 94% and reduced metabolic viability to 36% at 72 h, markedly outperforming free drugs and non-targeted controls. A single intravenous dose delivering 5 mg/kg BBR equivalent in CT26-bearing mice restricted tumor volume and mass to ~ 15% and ~ 13% of PBS controls, normalized splenomegaly four-fold, and produced pronounced decreases in Ki-67 and CD31 with increased TUNEL positivity, all without body-weight loss or organ pathology. These results demonstrate that AI-guided synergy scouting, ligand discovery and drug-allocation modelling can be seamlessly combined with modular nanocarrier engineering to generate a high-payload, CXCR4-targeted PTX/BBR therapy that delivers potent antitumor efficacy alongside excellent systemic tolerance, offering a transferable blueprint for next-generation combination nanomedicines. AI-guided nanomedicine CXCR4-targeted core–shell mesoporous silica nanoparticle liposome coating combination chemotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Combination chemotherapy, simultaneous administration two or more mechanistically distinct agents, has long been recognized as a s a means of improving clinical response and delaying chemoresistance in solid tumors. Typically, mono-chemotherapy, which relies on a single agent, is not sufficient to effectively inhibit tumor growth due to its one-dimensional action mechanism. This results in accelerating tumor relapse and the emergence of chemoresistance mutations.[ 1 – 3 ] In contrast, combination chemotherapy represents an attractive strategy for cancer treatment because the agents used have complementary mechanisms of action against cancer cells, which serve to strengthen and activate alternative pathways.[ 4 ] Additionally, combination chemotherapy not only achieves synergistically outcomes but also helps prevent the development of drug resistance in cancer cells.[ 5 – 7 ] In clinical and preclinical studies, combination chemotherapy has shown a more robust anti-cancer response and improved survival rates than mono-chemotherapy.[ 8 ] Nanoparticle-mediated drug delivery systems, such as inorganic nanoparticles, lipid nanoparticles, polymeric nanoparticles, and nano-scaffolds, are currently undergoing preclinical and clinical development for combination chemotherapy by delivering multiple drugs to various cancers, offering benefits over conventional anticancer therapy.[ 2 , 9 – 11 ] Nanoparticle-mediated drug delivery systems show passive cancer-targeting ability through the enhanced permeability and retention (EPR) effects, as well as active targeting capability by introducing receptor-specific ligands on the carriers.[ 12 – 14 ] Additionally, nanoparticles enable sustainable drug release, prevent degradation of agents, and minimize adverse effects, ultimately enhancing therapeutic efficacy.[ 15 , 16 ] Therefore, nanoparticle-mediated delivery of multiple therapeutic agents is an advanced approach due to the various physicochemical and pharmacological properties of nanoparticles for combination chemotherapy. Among various nanoparticle-mediated drug delivery systems, mesoporous silica nanoparticles (MSNs) are considered a promising delivery system for the development of cancer therapies. This is due to their many advantages, such as surface functionality, size tunability, high intrinsic stability, and high drug encapsulation efficiency resulting from their unique mesoporous structure.[ 17 ] While numerous published papers have successfully demonstrated the therapeutic effect of MSNs, very few in-vivo experiments have been found in the literature. This is primarily because of the low stability of MSNs in a biological medium, which leads to rapid accidental uptake by the reticuloendothelial system (RES).[ 18 – 20 ] The dispersity of MSNs has been difficult to maintain, often resulting in irreversible aggregation.[ 21 ] Moreover, the cytotoxicity of nanoscale MSN is significantly high at concentrations ≈ 25 µg/mL.[ 22 , 23 ] Additionally, MSNs lack specific cancer-targeting ability, which can lead to side effects. To address these critical issues, we employed two engineering strategies: (1) functionalization of MSNs through surface modification, and (2) coating functionalized MSNs (FMSNs) with liposomes. MSNs are surface-modified with various moieties (e.g., amino, phosphonate, and carboxylate groups) to prevent false positive/negative signals and maintain the MSNs well-dispersed in aqueous solution with minimal to no aggregation. The positively or negatively charged MSNs improve solubility in biological solutions through electrostatic interactions, resulting in high permeability.[ 24 , 25 ] Additionally, MSNs can be covalently bound to fluorescent dye molecules (e.g., fluorescein isothiocyanate [FITC] molecules) for bioimaging purposes.[ 26 ] The FMSNs can also be coated with liposomes that have opposite charges to enhance solubility and reduce cytotoxicity. Liposomes are a clinically proven delivery system as they exhibit non-immunogenicity, good biocompatibility, high delivery efficiency, and ability to encapsulate hydrophobic and hydrophilic drugs.[ 27 , 28 ] Furthermore, ligand-conjugated liposomes possess active targeting abilities towards cancer.[ 29 , 30 ] Therefore, liposome-coated FMSNs could be used as a promising multidrug delivery nanoplatform for cancer treatment. The selection of complementary drugs is crucial in combination chemotherapy, where distinct mechanisms maximize tumor eradication. Berberine (BBR) is an alkaloid with broad antitumor activity, including against colorectal cancer, mediated by inhibition of angiogenesis and proliferation through cell-cycle arrest, autophagy modulation, and apoptosis induction.[ 31 – 35 ] BBR preferentially accumulates in the mitochondria of cancer cells, where it dissipates the mitochondrial membrane potential, elevates reactive-oxygen species (ROS), and triggers mitochondrial permeability transition, culminating in apoptosis.[ 36 , 37 ] BBR further enforces AMP-activated protein kinase (AMPK)-driven metabolic reprogramming and blocks epithelial–mesenchymal transition in colorectal cancer cells, thereby suppressing tumor growth and metastasis.[ 38 ] While BBR demonstrates notable antitumor efficacy with relatively low toxicity, its ability to induce complete remission remains limited, necessitating combination strategies to enhance therapeutic outcomes. To rationally choose a co-therapeutic for BBR, we applied the AI-driven drug-synergy platform (MD-Syn) to screen FDA-approved oncology agents across multiple colorectal cancer cell models.[ 39 ] Paclitaxel (PTX) consistently emerged as the top synergistic candidate. PTX, a microtubule-stabilizing agent used in breast, ovarian, lung, and colorectal cancers,[ 40 – 43 ] not only arrests mitotic but also perturbs mitochondrial function to amplify apoptosis in malignant cells.[ 44 ] Its clinical efficacy, however, is often limited by multidrug resistance (MDR).[ 45 , 46 ] Because BBR acts through mitochondrial and metabolic pathways distinct from microtubule stabilization, the BBR/PTX combination offers complementary cytotoxic mechanisms while mitigating MDR, a premise supported by AI-predicted synergy and explored experimentally here.[ 47 ] In this study, we integrate (i) AI-guided ligand discovery and drug-synergy selection, (ii) AI-rational drug allocation (PTX in the lipid shell, BBR in the FMSN core), and (iii) liposome-coated silica engineering to create FMSN(BBR)-CXCR4BPL(PTX), which is a C-X-C chemokine receptor type 4 (CXCR4)-targeted core-shell nanocarrier loaded with BBR and PTX as a multidrug delivery system (Fig. 1 ). CXCR4 is overexpressed in colorectal carcinoma and correlates with metastasis and poor prognosis.[ 48 , 49 ] To confer CXCR4 selectivity we implemented a dual-AI peptide screen: CABS-dock and NeuroSNAP-AI [ 50 , 51 ]. We demonstrate that the platform (i) displays high dual-drug encapsulation efficiency, (ii) releases both agents in a sustained fashion, (iii) remains non-toxic up to 100 µg/mL, (iv) accumulates selectively in CXCR4-positive tumors, and (v) produces synergistic anti-proliferative and anti-migratory effects in-vitro and significant tumor regression in-vivo without systemic toxicity. Collectively, these findings showcase how AI-assisted selection of synergistic drug pairs and rational nanocarrier design can accelerate the development of next-generation, receptor-targeted combination nanotherapies. 2. Results and discussion 2.1. AI-guided selection of the CXCR4-binding peptide and fabrication of CXCR4BP-liposomes An AI-driven pipeline was first used to identify an optimal ligand for CXCR4 (Fig. 1 A). Flexible docking of SDF-1/CXCR4–derived sequences with CABS-dock ranked a 9-mer fragment of SDF-1 (Lys–Cys) highest.[ 50 , 52 – 54 ] Subsequent AlphaFold-multimer analysis with NeuroSNAP-AI returned an inter-protein TM-score (ipTM) of 0.95 and uniformly low predicted-aligned-error (PAE) values (< 10 Å) across the interface, while the predicted-distance-error (PDE) map displayed a tight blue–purple distribution (Fig. 2 B, 2 C, 2 D). These metrics indicate a high-confidence, well-defined binding pose; the peptide was therefore designated CXCR4-binding peptide (CXCR4BP). To anchor the ligand in a lipid membrane, CXCR4BP was conjugated to Maleimide-PEG(2000)-DSPE via a sulfhydryl–maleimide reaction (Fig. 2 A, right). LC/MS confirmed disappearance of the free peptide and maleimide peaks, giving a single product at the expected m/z (Fig. 2 E) with a coupling yield was 97.2 ± 2%. CXCR4BP-liposomes (CXCR4BPLs) were then prepared in a single thin-film hydration method. DSPC, DOTAP, cholesterol and the freshly synthesized CXCR4BP-lipid (67 : 8 : 20 : 5 mol %) were co-dissolved to form CXCR4BPL. For the controls, maleimide-PEG(2000)-DSPE was inserted into liposomes without CXCR4BP. Compared with peptide-free control liposomes (Mal-liposome), CXCR4BP-liposomes whose mean diameter (122 ± 4 nm) was indistinguishable from maleimide control liposomes (119 ± 5 nm; Fig. 2 F). In contrast, the ζ-potential shifted from 12.3 ± 3.2 mV to 26.1 ± 5.9 mV after peptide insertion, consistent with surface display of the mildly cationic ligand (Fig. 2 G). A bicinchoninic-acid (BCA) assay quantified 112.6 ± 6.2 µg peptide per mg lipid, confirming successful and reproducible incorporation (Fig. 2 H). Collectively, these results validate (i) the computationally selected CXCR4BP as a high-affinity ligand, (ii) its efficient chemical linkage to DSPE lipid, and (iii) the generation of size-conserved liposomes bearing a peptide-rich corona, laying the foundation for a subsequent core–shell nanocarrier assembly. 2.2. Synthesis and physicochemical characterization of phosphonate/FITC-functionalized MSNs (FMSNs) MSNs were produced by a base-catalyzed sol–gel reaction and subsequently co-functionalized with 3-trihydroxysilyl­propyl­methyl­phosphonate (THMP) and FITC-APTMS (see Methods). FITC offers bioimaging function, while phosphonate confers high solubility in aqueous solution and cationic liposome coating. The broad halo at 2θ ≈ 22° in the X-ray diffractometer (XRD) profile confirms the amorphous silica framework is retained after surface modification (Fig. 3 A).[ 55 , 56 ] The field emission-transmission electron microscope (FE-TEM) reveals well-defined and radially aligned mesopores with an average particle diameter of ~ 150 nm (Fig. 3 B, left ). Scanning transmission electron microscopy with energy dispersive spectroscopy (STEM-EDS) elemental maps display that phosphorus (magenta) is homogeneously distributed throughout the silica matrix (cyan) (Fig. 3 B, right ), while the corresponding EDS spectrum exhibits clear Si and P peaks (Fig. 3 D), further confirming surface modification. The exact amount of phosphorus was identified as 6.52 wt% (Table 1 ). These results confirmed that the P element was successfully functionalized onto the MSNs. Fourier transform infrared spectrophotometer (FTIR) comparison of pristine MSNs and functionalized FMSNs (Fig. 3 E) provides further evidence of successful phosphonate functionalization. In addition to the common Si-O-Si bands at 1080 and 810 cm − 1 ,[ 57 ] the FMSN trace displays a new P = O stretching band at ~ 1230 cm − 1 (inset), whereas the parent MSN lacks these features, indicative of successful THMP attachment on FMSNs. FITC labelling endows the particles with bright green fluorescence, and the intensity of which increased linearly with dispersion concentration and exhibited a maximum at 535 nm (Fig. 3 F, inset photograph). Collectively, these results demonstrate that the dual functionalization strategy preserves the ordered mesoporous architecture while imparting aqueous dispersibility, traceable fluorescence and a phosphonate-rich surface for subsequent liposome coating and drug loading. Table 1 STEM-EDS elemental composition of phosphonate/FITC-functionalized mesoporous silica nanoparticles (FMSNs). Quantitative analysis confirms successful phosphonate grafting, with phosphorus accounting for 6.52 wt % (5.95 at %). Element Line Type k Factor k Factor type Absorption Correction Wt% Wt% Sigma Atomic% N K series 1.807 Theoretical 1.00 0.00 0.00 0.00 Si K series 1.000 Theoretical 1.00 93.48 1.83 94.05 P K series 1.036 Theoretical 1.00 6.52 1.83 5.95 Total: 100.00 100.00 2.3. CXCR4BPL coating on FMSN and characterization of FMSN-CXCR4BPL The self-assembly was initiated by hydrating a thin film of CXCR4BPL with FMSN using probe-sonication to facilitate electrostatic interaction between the negatively charged FMSN and cationic CXCR4BPL.[ 58 ] BCA assay, FE-TEM with STEM-EDS mapping, field-emission scanning electron microscopy (FE-SEM), and Confocal laser-scanning microscopy (CLSM) were used to confirm the liposome coating. Following the liposome coating, FMSN-CXCR4BPLs exhibited a significantly higher protein content than FMSNs (Fig. 4 A), attributed to the peptide portion of CXCR4BPLs. The FE-TEM combined with STEM-EDS mapping visualizes a clear core–shell architecture of FMSN-CXCRBPL (Fig. 4 B, Table 2 ). Silicon and phosphorus signals co-localize in the FMSN cores, whereas carbon is confined to the liposome shells within the same regions of the FMSN-CXCR4BPL particles. FE-SEM highlights the resulting surface transformation from a smooth silica texture to a blurred, uniformly coated surface indicative of the surrounding lipid layer (Fig. 4 C). In addition, CLSM shows co-localization of FITC-FMSN (green) and DiI-labelled CXCR4BP (red) signals, confirming that each FMSN core is uniformly enveloped by the lipid shell (Fig. 4 D). We further characterized FMSN, FMSN-CXCR4BPL, and CXCR4BPL. Dynamic-light-scattering (DLS) analysis indicates that the hydrodynamic diameter of FMSN (≈ 130 nm) is maintained after coating (Fig. 4 E), confirming that liposome coating does not appreciably change particle size. As expected, the ζ-potential shifts from − 7.3 ± 1.5 mV to + 8.0 ± 2.4 mV due to the positive charge of peptides (Fig. 4 F). The core–shell nanocarrier is colloidally stable in PBS for at least 7 days, with no significant change in PDI (Fig. 4 G) or average size (Fig. 4 H). Table 2 STEM-EDS elemental composition of liposome-coated nanoparticles (FMSN-CXCR4BPL). The marked increase in carbon (~ 39 wt %) relative to bare FMSNs, together with the residual silicon and trace phosphorus from the core, confirms the presence of an organic lipid shell surrounding the silica core. Element Line Type k Factor k Factor type Absorption Correction Wt% Wt% Sigma Atomic% C K series 3.115 Theoretical 1.00 39.49 0.26 51.45 O K series 1.455 Theoretical 1.00 35.28 0.21 34.51 Si K series 1.000 Theoretical 1.00 24.80 0.17 13.82 P K series 1.036 Theoretical 1.00 0.43 0.04 0.22 Total: 100.00 100.00 2.4. Synergy screening and BBR/PTX co-loading of FMSN-CXCR4BPL BBR was chosen as the anchor drug because it triggers cell-cycle arrest, AMP-activated protein kinase (AMPK)-driven metabolic reprogramming, and inhibition of epithelial–mesenchymal transition in colorectal cancer cells, thereby suppressing tumor growth and metastasis.[ 38 ] While BBR exhibits broad antitumor activity and relatively low toxicity, its monotherapeutic potential remains limited due to incomplete tumor eradication and risk of recurrence, necessitating a rationally selected co-therapeutic agent to enhance overall efficacy. To identify a truly cooperative drug, we employed the MD-Syn AI platform to screen BBR against 38 FDA-approved oncology agents across six human colorectal-cancer cell lines (HCT116, HT29, LoVo, RKO, SW620, SW837),[ 39 ] which served as surrogates in lieu of a CT26 murine model. PTX emerged as the compound with a synergy-probability > 0.9999 in every cell line (grand mean = 0.999999852; Fig. 4 E), justifying its selection for combination therapy. Prior to formulation, we employed the open-source FormulationAI drug encapsulation module.[ 59 ] The model predicted encapsulation efficiencies of 52.56% (BBR in liposome) and 68.65% (PTX in liposome). According to these AI-guided results, we decided to encapsulate PTX in CXCR4BPL part and load BBR in FMSN. After one-pot self-assembly, the resulting core–shell nanocarrier achieved BBR EE = 78.8 ± 1.9% and PTX EE = 75.2 ± 2.4% (Fig. 4 J), suggesting an optimized loading strategy for this core-shell loading combination. Additionally, the release kinetics of BBR and PTX from FMSN(BBR)-CXCR4BPL(PTX) demonstrated sustained release over a 72-h period in PBS (pH 7.4, 37°C), indicating prolonged drug availability that could enhance targeted cancer cells treatment while maintaining effective therapeutic levels (Fig. 4 K). These results support the rationale for combining BBR with PTX, as the core–shell architecture not only accommodates both drugs at high loading capacity but also ensures their sustained co-delivery, a crucial factor in achieving the predicted synergistic antitumor effects. 2.5. In-vitro biocompatibility of FMSN-CXCR4BPL The in-vitro biocompatibility of bare FMSN and lipid-coated FMSN-CXCR4BPL was assessed using a live/dead cell assay and CCK-8 assay on CT26 colon-carcinoma cells and human adipose-derived MSCs (hASC) at various concentrations after 48 h incubation (Fig. 5 ). As shown in Fig. 5 A and 5 B, CT26 and hASC exposed to bare FMSN at 100 µg/mL displayed an increase in dead cells (red fluorescent), whereas little or no red signal was detected at 1 µg/mL and 10 µg/mL. In contrast, cells treated with FMSN-CXCR4BPL retained an almost exclusively green fluorescence pattern even at 100 µg/mL, indicating minimal membrane damage. The lipid shell effectively masks the potential toxicity at high concentration of the FMSN cores (Fig. 5 A, 5 B). These observations were corroborated by the CCK-8 assay results. For CT26, viability fell to 74.6 ± 1.5% with bare FMSN at 100 µg/mL but remained 93.9 ± 9.5% with FMSN-CXCR4BPL (Fig. 5 C). hASC showed a similar trend (bare FMSN 77.1 ± 3.8%; coated FMSN-CXCR4BPL 97.3 ± 4.5%, Fig. 5 D). No significant difference was observed between the two formulations at 1 µg/mL or 10 µg/mL. These results agree with previous reports that uncoated MSNs become dose-limiting above ≈ 25 µg/mL owing to exposed silanol groups.[ 22 , 23 ] The phospholipid shell passivates reactive silanols, thereby suppressing membrane disruption and improving serum compatibility. Because the CXCR4BP is present at only 0.5 mol % of total lipid, the shell itself is essentially non-toxic while conferring receptor-mediated targeting capability. Collectively, the lipid corona transforms a dose-limited inorganic carrier into a biocompatible platform that remains safe at 100 µg/mL, which is a concentration exceeding typical therapeutic doses used in subsequent in-vivo studies. 2.6. CXCR4-mediated cancer-targeting ability in-vitro and in-vivo To evaluate whether CXCR4BPs enhance tumor selectivity, we compared bare FMSN with FMSN-CXCR4BPL in CXCR4-overexpressing CT26 colon-carcinoma cells and in CT26-tumor-bearing mice (Fig. 6 ). CLSM showed time-dependent internalization of both formulations, but FMSN-CXCR4BPL produced markedly stronger intracellular fluorescence from 3 h onward (green) than bare FMSN (Fig. 6 A). ImageJ analysis confirmed that the percentage of nanoparticle-positive CT26 cells rose to 18.9 ± 0.3% for FMSN-CXCR4BPL versus 0.7 ± 0.1% for FMSN after 3 h (Fig. 6 B). Flow cytometry analysis was conducted on CT26 cells to substantiate these findings with additional quantitative support. Flow cytometry histograms echoed this trend. FITC-positive populations appeared as early as 0.5 h and reached a three-fold higher mean fluorescence intensity for FMSN-CXCR4BPL at 3 h (Fig. 6 C, 6 D). These data demonstrate efficient CXCR4-mediated attachment and internalization of FMSN-CXCR4BPL in-vitro . To evaluation of CXCR4-mediated tumor-targeting ability of FMSN-CXCR4BP, IR780-loaded FMSNs and FMSN-CXCR4BPLs were injected i.v. (0.5 mg/kg IR780) to CT26-tumor-bearing BALB/c mice. Whole-body imaging revealed progressive tumor-specific fluorescence, peaking at 24–48 h. Signal in the FMSN-CXCR4BPL group was consistently higher than in the FMSN group, indicating improved targeting and/or retention (Fig. 6 E). Ex-vivo fluorescence imaging 48 h post-injection showed the strongest fluorescence in tumor relative to other organs (Fig. 6 F). Quantification confirmed a three-fold higher radiant efficiency in tumors treated with FMSN-CXCR4BPL compared with bare FMSN (p < 0.0001; Fig. 6 G). Together, the CLSM/flow cytometry in-vitro data and the in-vivo imaging results establish that the CXCR4-binding lipid corona confers robust and selective targeting to CXCR4-positive cancer cells, leading to enhanced tumor accumulation without off-target organ uptake. 2.7. In-vitro anticancer efficacy of FMSN(BBR)-CXCR4BPL(PTX) The targeted core–shell nanocarrier (FMSN(BBR)-CXCR4BPL(PTX)) was compared with a free BBR/PTX mixture and a non-targeted silica formulation, FMSN(BBR/PTX). Because CXCR4 signaling is a key driver of tumor dissemination, we first assessed cell migration, a prerequisite for metastasis, alongside direct cytotoxicity.[ 60 ] In a scratch-wound assay (Fig. 7 A, 7 B), control monolayers almost completely closed > 97% of the gap within 24 h, and free dual-drug mixture (BBR/PTX) reduced closure only modestly. Non-targeted FMSN(BBR/PTX) slowed migration further, yet a continuous front was still visible. In contrast, FMSN(BBR)-CXCR4BPL(PTX) preserved 93.6 ± 2.9% of the initial gap after 24 h, significantly higher than all other groups (Fig. 7 B). Metabolic activity measured by CCK-8 assay followed the same drug-dose–dependent trend (Fig. 7 C). After 72 h, cancer cell viability fell to 36.2 ± 1.9% with the targeted nanocarrier, whereas free drugs and non-targeted particles left 81.1 ± 4.7% and 68.9 ± 20.1% viable cancer cells, respectively. Live/dead staining corroborated these data (Fig. 7 D). Extensive EthD-1 (red) fluorescence was evident only in the FMSN(BBR)-CXCR4BPL(PTX) group at 72 h. Thus, by combining CXCR4-specific delivery with complementary mitochondrial (BBR) and mitotic (PTX) mechanisms, the core–shell nanocarrier not only kills CT26 cancer cells more efficiently but also blocks the migratory behavior that underlies metastasis, highlighting its dual anti-cancer potential. 2.8. In-vivo antitumor efficacy of FMSN(BBR)-CXCR4BPL(PTX) Building on promising in-vitro cytotoxicity and significant tumor accumulation, we evaluated the antitumor efficacy of FSMN(BBR/PTX) and FMSN(BBR)-CXCR4BPL(PTX) nanoparticles in a CT26 tumor-bearing mice model. When tumors reached approximately 100 mm 3 in volume, mice were randomly assigned to six treatment groups: PBS (control), free BBR, free PTX, a free BBR/PTX combination, FSMN(BBR/PTX), and FMSN(BBR)-CXCR4BPL(PTX). A single i.v. administration of the CXCR4-targeted core–shell nanocarrier produced the most pronounced tumor control among the six treatment arms. Tumors in the PBS control group expanded steadily, reaching ≈ 1500 mm³ by day 21, whereas growth was only modestly delayed by free BBR or PTX alone. A combined injection of the two free drugs and the non-targeted FMSN(BBR/PTX) did slow tumor growth, yet a clear expansion persisted. In contrast, FMSN(BBR)-CXCR4BPL(PTX) virtually halted tumor enlargement, limiting the final volume to 14.6 ± 4.8% of the PBS group (Fig. 8 A). Tumor growth data were corroborated by endpoint tumor weights. the targeted nanocarrier reduced mean tumor mass from 4.67 ± 0.3 g in PBS controls to 0.62 ± 0.1 g, i.e. ≈ 13.3 ± 2% of the PBS control value (Fig. 8 B). This weight reduction parallels the volumetric inhibition, and the excised tumors displayed the same hierarchy of mass (Fig. 8 C). The results highlight the superior in-vivo potency of FMSN(BBR)-CXCR4BPL(PTX) over free drugs or the non-targeted formulation. Systemic tolerance was excellent, since no significant body-weight changes were observed in any group over 21 days (Fig. 8 D), and H&E examination revealed no histopathological damage to heart, liver, spleen, lung, or kidney (Fig. 8 G). Spleen weight served as an additional systemic read-out. In this model, tumor-driven inflammation produces pronounced splenomegaly.[ 61 ] Notably, untreated mice exhibited marked splenomegaly (876.2 ± 68.9 mg), whereas the targeted formulation normalized spleen weight to 214.3 ± 55.1 mg, roughly a four-fold reduction (Fig. 8 E, 8 F). The reversal of splenomegaly therefore indicates that the targeted therapy not only shrinks the primary tumor but also alleviates tumor-induced systemic immune dysregulation, further underscoring its favorable safety–efficacy profile. To further confirm the enhanced antitumor activity of FMSN(BBR)-CXCR4BPL(PTX), particularly in relation to cell proliferation, immunohistochemistry staining was performed. Ki-67 fluorescence fell to 7.0 ± 5.2% of control, indicating strong proliferation arrest (Fig. 8 H, 8 K). A TUNEL assay revealed a 6-fold rise in apoptotic nuclei relative to PBS (Fig. 8 I, 8 L), while CD31 labelling showed a one-tenth drop in micro-vessel density, confirming potent anti-angiogenic action (Fig. 8 J, 8 M). Together, these results demonstrate that CXCR4-directed co-delivery of BBR and PTX achieves multi-modal tumor suppression, curbing proliferation, inducing apoptosis, and starving neo-vasculature without systemic toxicity, thereby validating the therapeutic promise of the FMSN(BBR)-CXCR4BPL(PTX) nanocarrier in-vivo . 3. Conclusion By chaining three orthogonal AI modules, we established a rapid, data-driven route to a potent, receptor-targeted combination chemotherapy for colorectal cancer. (i) MD-Syn screening of 38 FDA-approved oncology drugs in six human colorectal-cancer cell lines (HCT116, HT29, LoVo, RKO, SW620, SW837) identified PTX as the compound with a > 0.9999 probability of synergy with BBR, nominating the PTX/BBR pair for formulation. (ii) A dual-AI peptide pipeline, flexible docking with CABS-dock followed by AlphaFold-multimer scoring in NeuroSNAP-AI, pinpointed a high-affinity 9-mer SDF-1 fragment (CXCR4BP; ipTM = 0.95) for selective targeting of the overexpressed CXCR4 receptor in colorectal tumors. (iii) FormulationAI predicted that loading BBR into FMSN cores and PTX into a CXCR4BPL shells. One-pot self-assembly yielded FMSN(BBR)-CXCR4BPL(PTX) with experimental entrapment efficiencies of 78.8 ± 1.9% (BBR) and 75.2 ± 2.4% (PTX), together with sustained co-release over 72 h. The ~ 130 nm core–shell nanocarrier curtailed scratch-wound closure by ≈ 94% and reduced metabolic viability to 36% in CXCR4-positive CT26 cells, vastly outperforming free drugs and non-targeted controls. In CT26-bearing mice, a single intravenous dose (5 mg/kg BBR equivalent) limited tumor volume and mass to ~ 15% and ~ 13% of PBS controls, normalized splenomegaly four-fold, and triggered strong anti-proliferative (↓Ki-67), pro-apoptotic (↑TUNEL), and anti-angiogenic (↓CD31) responses without body-weight loss or histopathological injury. Collectively, these results demonstrate that AI-guided synergy scouting, ligand discovery, and drug-placement optimization, combined with modular nanocarrier engineering, can deliver high-payload, receptor-targeted combination therapeutics that couple potent efficacy with systemic tolerance. The workflow is readily transferable to other ligand–receptor axes and drug pairs, offering a general blueprint for accelerating next-generation anticancer nanomedicines. 4. Experimental Section Cell culture Colon carcinoma cells (CT26) and human adipose-derived stem cells (hASCs) were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. Cell lines were incubated at 37°C in a 5% CO 2 humidified atmosphere. Preparation of phosphonate/fluorescence-functionalized MSNs (FMSNs) and loading of BBR The FMSNs were synthesized using a base-catalyzed sol-gel method at high temperatures, modifying previous protocols.[ 62 – 64 ] The FITC-labeled MSNs were prepared by reacting 1.4 mmol of FITC with 1.2 mmol of (3-aminopropyl)trimethoxy silane (APTMS) in ethanol under an argon atmosphere for 2 h. The FITC-APTMS product was then added to a co-condensation reaction of 5.0 mL of tetraethyl orthosilicate (TEOS), 1.0 g of hexadecyltrimethylammonium bromide (CTAB) in a mixture of 480 mL of distilled water, and 3.5 mL of 2 М sodium hydroxide. After 15 min of stirring at 80 ℃, 1.3 mL of 3-trihydroxysilylpropylmethylphosphonate (THMP) was added dropwise to the mixture. The reaction mixture was vigorously stirred at 80 ℃ for 2 h. Once the reaction was complete, the solution was cooled to ambient temperature, and the FITC-labeled nanoparticles were filtered and washed thoroughly with methanol using a fritted funnel under vacuum for 24 h. To remove the CTAB surfactants, 1.0 g of the nanoparticles were dissolved in a mixture of 100 mL of methanol and 1.0 mL of 37% hydrochloric acid. After refluxing the solution for 24 h, the nanoparticles were filtered and washed thoroughly with methanol through a fritted funnel under vacuum for 24 h. FMSN characterization was performed using an X-ray diffractometer (XRD). The validation of fluorescence of FMSN was determined using a microplate reader (SynergyTM H1, BioTek Instruments Inc.) by exciting the samples at 480 nm and measuring the emission spectra between 500 and 700 nm. Phosphonate-surface modification was verified using a Fourier transform infrared spectrophotometer (FTIR) and a field emission-transmission electron microscope (FE-TEM) coupled with energy-dispersive X-ray spectroscopy (EDS). To load drug molecules into the pores of the particles, the FMSNs were soaked in a concentrated solution containing the drugs. Typically, 100 mg of FMSNs were gently stirred in a solution containing 100 mg of BBR and ethanol: distilled water (7:3 v/v) solution for 48 h. After stirring, the mixture was centrifuged at 23,000 g for 10 min several times, and the supernatant was removed. The FMSN(BBR)s were then lyophilized. Preparation and characterization of peptide-conjugated lipids Peptides that bind to CXCR4 and contain an additional cysteine at the N-terminus was conjugated with Maleimide-PEG(2000)-DSPE through a sulfhydryl-maleimide coupling reaction.[ 65 ] In brief, Maleimide-PEG(2000)-DSPE was first diluted in chloroform and then rotary evaporated at 37 ℃ and 120 rpm to create a thin film, which was then hydrated with pure water at 37°C. The peptides were dissolved in 0.1 М phosphate-buffered solutions (PBS) and mixed with Maleimide-PEG(2000)-DSPE (peptides/Maleimide-PEG(2000)-DSPE molar ratio of 1.2:1) and left to react overnight under argon gas at room temperature. The resulting mixture was then placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa for 48 h to remove the residual free peptides. After dialysis, the solution was dried through lyophilization. The peptide conjugation was analyzed using liquid chromatography-mass spectrometry (LC/MS) (Shimadzu, Japan) and bicinchoninic-acid (BCA) assay. Preparation of FMSN(BBR)-CXCR4BPL(PTX) The conventional thin-film hydration method was applied to prepare PTX-loaded liposomes[ 66 , 67 ] composed of 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane, cholesterol, and CXCR4BP-conjugated lipids (CXCR4BP-lipids) (67:8:20:5, molar ratio). Briefly, the lipids (lipid-to-FMSN mass ratio 10:1) and PTX (5%, wt/wt) were dissolved in chloroform, and the organic solvent was evaporated under reduced pressure at 70 ℃ using a hot drying oven for 2 h to form a thin film. The thin film was then hydrated with distilled water at 60 ℃ for 10 min. Following hydration, the lipid mixture was mixed with FMSN(BBR) solution and subjected to be stirred for 24 h. The mixture was then sonicated in an ice-water bath using probe sonication (on 2 s/off 2 s, 20 min, 26% amplitude). To remove any unencapsulated drugs, the FMSN(BBR)-CXCR4BPL(PTX) were centrifuged at 23,000 g for 10 min three times. Finally, the nanocarriers were dried through lyophilization. DiI-labeled CXCR4BPLs were prepared using the same methods, except that DiI was added before formation of the thin film. Physicochemical characterization of FMSN-CXCR4BPL The mean particle sizes and nanoparticle number concentration of FMSN, FMSN-CXCR4BPL, and CXCR4BPL were measured using nanoparticle tracking analysis (NTA) with a NanoSight NS300 (Malvern Instruments Ltd., Worcestershire, UK, camera level = 12, detection threshold = 5). The polydispersity indexes (PDIs) and zeta potentials (surface charges) of FMSN, FMSN-CXCR4BPL, and CXCR4BPL were determined via dynamic light scattering (DLS) using a Zetasizer Nano ZS system (Malvern Instruments Ltd., Worcestershire, UK). The stability of the core-shell nanocarriers was assessed by monitoring changes in size and PDI over 7 d. Morphology and composition were confirmed through FE-TEM coupled with EDS and field-emission scanning electron microscopy (FE-SEM). Additionally, CLSM was used to validate liposome coating and drug loading. Protein quantification of FMSN and FMSN-CXCR4BPL was performed using the bicinchoninic acid (BCA) assay. BBR or PTX encapsulation efficiency and release profiles The amount of loaded BBR or PTX was calculated using a microplate reader and high-performance liquid chromatography (HPLC, Agilent, CA, USA). To determine the amount of BBR or PTX in the core-shell nanocarrier, the FMSN-CXCR4BPL was processed with 1N HCl using a bath sonicator (100 W). Encapsulation efficiency (EE) was then calculated using the following equation: $$\:Drug\:encapsulation\:\left(\%\right)=\left(\:\frac{Amount\:of\:\:drug\:encapsulated\:in\:FMSN-CXCR4BPL}{Total\:amount\:of\:drug\:}\right)\times\:100\:$$ 1 To measure the release profile of BBR or PTX, 400 µL of FMSN(BBR)-CXCR4BPL(PTX) was dialyzed against 14 mL of PBS (pH 7.4) with constant shaking (100 rpm) at 37°C. At different time intervals, 1 mL of the dialysis buffer solution was aliquoted for measurement and then replaced with an equal volume of fresh medium. The amount of released BBR or PTX was determined using a microplate reader or HPLC to quantify the concentration of each drug. Biocompatibility of FMSN-CXCR4BPL The LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen™, USA) was used to assess the cytotoxicity of FMSN and FMSN-CXCR4BPL via fluorescence microscopy. CT26 and hASC cells were seeded at a density of 3 × 10 4 cells/mL in 24-well plates. After 24 h, the cells were exposed to FMSN or FMSN-CXCR4BPL (1, 10, and 100 µg/mL) in the cell-culture medium for 48 h. Following the 48 h incubation, the cells were treated with a live/dead solution (5 µL of calcein AM for live cells and 20 µL of ethidium homodimer (EthD-1) for dead cells in 10 mL of cell medium) for 30 min before observation under fluorescence microscopy. To evaluate the biocompatibility of FMSN and FMSN-CXCR4BPL, a cell counting kit-8 (CCK-8, DOJINDO, Japan) assay was performed. CT26 and hASC cells were seeded at a density of 1 × 10 4 cells/mL in 96-well plates and incubated for 24 h. The cells were then exposed to FMSN or FMSN-CXCR4BPL (1, 10, and 100 µg/mL) for 48 h. After the 48 h incubation, the cell-culture medium was replaced with a 10% CCK-8 solution in each well and incubated at 37°C for an additional 2 h. The absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated based on the relative absorbance compared to the control absorbance. The use of hASCs was approved by the Institutional Review Board of Chung-Ang University Hospital (Approval No. 2151-005-463) and conducted in compliance with the principles outlined in the Declaration of Helsinki. In-vitro cellular uptake of FMSN-CXCR4BPL for validation of cancer-cell-targeting ability via confocal laser-scanning microscopy (CLSM) and flow cytometry The cancer cell targeting ability was determined via CLSM. CT26 cells were seeded (1 × 10 4 cells/chamber) on 4-well glass-chamber slides (SPL, Cell Culture Slide) in cell-culture medium. After 24 h, the cells were treated with fresh medium containing either 1×10 8 FMSN or 1×10 8 FMSN-CXCR4BPL for 0.5, 1, 3, and 5 h at 37°C. Following incubation, the chambers were washed at least three times with PBS (pH 7.4). The cells were then incubated in PBS with rhodamine phalloidin for 2 h for cytoplasm staining and DAPI for 3 min for nuclear staining. After washing several times with PBS, the chambers were mounted and examined using a Confocal Zeiss LSM 900 microscope, with ZEISS ZEN lite software used for setup. Flow cytometry was used to determine the cancer cell targeting ability. CT26 cells (2 × 10 5 cells/mL) were cultured overnight at 37°C under a 5% CO 2 atmosphere. The cellular uptake of FMSN and FMSN-CXCR4BPL was measured by incubating the cells with 1 × 10 8 FMSN and FMSN-CXCR4BPL in cell-culture medium for 0.5, 1, 3, and 5 h. Following incubation, the cells were washed twice with PBS, and the fluorescence was analyzed using a BD Accuri C6 Plus flow cytometer (BD Bioscience, USA). Cells cultured in the absence of the core-shell nanocarrier were used as the control. The experiments were repeated at least three times. In-vitro cancer migration and cytotoxicity test of FMSN(BBR)-CXCR4BPL(PTX) for validation of anti-cancer effect. Cell migration was assessed using a scratch assay. CT26 colon carcinoma cells were seeded at 8 × 10 6 cells/mL in a 60-mm dish and incubated at 37°C for 24 h to form a confluent monolayer. Subsequently, scratches were made using a 10 µL micropipette tip. The scratched cell monolayers were then treated with cell-culture medium including BBR (1 µg/mL)/ PTX (0.4 µg/mL), FMSN containing BBR (1 µg/mL)/ PTX (0.4 µg/mL), and FMSN-CXCR4BPL containing BBR (1 µg/mL)/ PTX (0.4 µg/mL). After 0, 6, 12, and 24 h cell culture, images of the scratch areas were captured using phase-contrast microscopy and analyzed using Image J software. The extent of cell migration was determined using the following equation: $$\:Relative\:gap\:area=\left(\:\frac{gap\:area\:at\:the\:indicated\:time\:point}{gap\:area\:at\:0\:h\:}\right)$$ 2 Quantitative cytotoxicity was determined using a CCK-8 assay in 96-well plates. CT26 cells were seeded at a concentration of 7 × 10 3 cells/mL and incubated for 24 h. The cells were then treated with BBR (1 µg/mL)/ PTX (0.4 µg/mL), FMSN containing BBR (1 µg/mL)/ PTX (0.4 µg/mL), and FMSN-CXCR4BPL containing BBR (1 µg/mL)/ PTX (0.4 µg/mL) for 24, 48, and 72 h. At each time point, 10% CCK-8 solution was added to each well and incubated at 37°C for an additional 2 h. The absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated as the relative absorbance compared to the control absorbance. Qualitative cytotoxicity of each group was assessed using the LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen™, USA). CT26 cells at a density of 3 × 10 4 cells/mL were seeded in 24-well plates. After 24 h of culture, the cells were treated with BBR (1 µg/mL)/ PTX (0.4 µg/mL), FMSN containing BBR (1 µg/mL)/ PTX (0.4 µg/mL), and FMSN-CXCR4BPL containing BBR (1 µg/mL)/ PTX (0.4 µg/mL) for 24, 48, and 72 h. At each time point, the CT26 cells were incubated with the live/dead solution for 30 min before fluorescence microscopy. In-vivo evaluation of tumor targetability and biodistribution Balb/c mice were subcutaneously (s.c.) injected with 1 × 10 6 CT26 cells into the right flank to induce tumor formation. Once the tumors reached an approximate volume of 100 mm³, the mice received intravenous (i.v.) injections of FSMN and FMSN-CXCR4BPL loaded with IR780 for imaging (IR780 dose: 0.5 mg/kg). The biodistribution and tumor-targeting efficiency of FMSN-CXCR4BPL were monitored over time using optical imaging, with the assistance of the fluorescence-labeled organism bio-imaging system (FOBI; Neo-Science, Gyeonggi, Korea). Near-infrared fluorescence (NIRF) intensity measurements were used to evaluate the tumor accumulation of FMSN-CXCR4BPL. After 48 h post-injection, the mice were euthanized, and both tumors and major organs (liver, lungs, spleen, heart, and kidneys) were harvested. NIRF images of the excised tissues were then analyzed to determine the distribution and tumor accumulation of the treatment. All animal experiments were conducted in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines of Chonnam National University, South Korea (Approval No. CNU IACUC-H-2024-23). In-vivo antitumor activity BALB/c mice were s.c. injected with 1 × 10 6 CT26 cells into the right flank to establish tumors. Once the tumors reached an approximate volume of 100 mm³, the mice were randomly assigned to one of six groups (four mice per group): PBS (control), free BBR, free PTX, a combination of BBR/PTX, FSMN(BBR/PTX), and FMSN(BBR)-CXCR4BPL(PTX). Treatments were administered intravenously, with each mouse receiving 100 µL of either PBS or the prepared nanoparticle formulations, corresponding to a BBR dose of 5 mg/kg, on day 0. Tumor size was measured every 2 d using Vernier calipers, and tumor volume was calculated using the equation: $$\:length\times\:{width}^{2}\times\:\frac{1}{2}$$ 3 Body weight was recorded at each measurement point. On day 21 post-treatment, the mice were euthanized, and tumors, along with major organs (heart, liver, spleen, lungs, and kidneys), were harvested. Tumors and major organs were fixed in 10% neutral buffered formalin, then paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). The H&E-stained sections were subsequently examined under an inverted light microscope. Immunohistochemical analysis Following deparaffinization, tissue slides underwent antigen retrieval through heat-mediated incubation in citrate buffer (pH 6.0) at 60°C for 15 min. After retrieval, sections were blocked with 5% bovine serum albumin (BSA) to prevent non-specific binding. The slides were then incubated overnight at 4°C with primary antibodies specific for Ki67 (catalog #ab15580) and CD31 (catalog #cell signalling 77699S), diluted in a blocking buffer containing 1% BSA in PBST (phosphate-buffered saline with Tween 20). Following primary antibody incubation, the sections were washed three times with PBST and subsequently incubated for 1 h at room temperature with secondary antibodies in PBST. Fluorescence images were acquired using a CLSM. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay Tumor tissue sections were prepared for TUNEL assay to assess apoptotic cell death. The sections were first deparaffinized to remove embedding materials. Following deparaffinization, staining was conducted using the Dead End™ Fluorometric TUNEL System (Promega) following the manufacturer’s instructions. After staining, fluorescence images were acquired and analyzed with a CLSM to evaluate the extent of apoptosis in the tumor tissues. Statistical analysis Experimental data are presented as means ± standard error of the mean (SEM). Statistical analyses were conducted using unpaired Student’s t-test for comparisons between two groups, one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for comparisons involving more than three groups. For comparisons of two independent variables, two-way ANOVA was used, followed by Tukey's post-hoc test. Statistical significance was determined with the following criteria: * p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 . All analyses were performed using GraphPad Prism 6 (GraphPad Software). Declarations Acknowledgments This research was supported by the National Research Foundation of Korea (NRF) funded by Ministry of Science and ICT (No. RS-2024-00449435 and No. 2020R1A2C2005620). Conflict of interest disclosure The authors declare no conflicts of interest. Author contributions Y.J. conceived and designed the project, performed all AI-driven tasks, synthesized all materials, performed physicochemical characterisation, executed and analyzed all in-vitro assays, curated the data, prepared the figures and tables, and wrote the manuscript. A.B. led the in-vivo studies, with technical assistance from S.C and A.V. J.J.M. provided feedback throughout the study. I.K.P. and H.P. supervised the research, secured funding, and contributed to project administration. All authors approved the final version for publication. Ethics approval statement All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee Chonnam National University (CNU IACUC-H-2024-23), South Korea. References Zimmermann GR, Lehar J, Keith CTJDdt: Multi-target therapeutics: when the whole is greater than the sum of the parts. Drug discovery today 2007, 12: 34-42. Pushpalatha R, Selvamuthukumar S, Kilimozhi DJJoDDS, Technology: Nanocarrier mediated combination drug delivery for chemotherapy–A review. Journal of Drug Delivery Science and Technology 2017, 39: 362-371. Kolishetti N, Dhar S, Valencia PM, Lin LQ, Karnik R, Lippard SJ, Langer R, Farokhzad OCJPotNAoS: Engineering of self-assembled nanoparticle platform for precisely controlled combination drug therapy. Proceedings of the National Academy of Sciences 2010, 107: 17939-17944. Hu C-MJ, Zhang LJBp: Nanoparticle-based combination therapy toward overcoming drug resistance in cancer. Biochemical pharmacology 2012, 83: 1104-1111. Gurunathan S, Kang M-H, Qasim M, Kim J-HJIjoms: Nanoparticle-mediated combination therapy: Two-in-one approach for cancer. International journal of molecular sciences 2018, 19: 3264. Devita Jr VT, Young RC, Canellos GPJC: Combination versus single agent chemotherapy: a review of the basis for selection of drug treatment of cancer. Cancer 1975, 35: 98-110. Jia J, Zhu F, Ma X, Cao ZW, Li YX, Chen YZJNrDd: Mechanisms of drug combinations: interaction and network perspectives. Nature reviews Drug discovery 2009, 8: 111-128. Xu J, Jin B, Ren Z, Lou Y, Zhou Z, Yang Q, Han BJPo: Chemotherapy plus erlotinib versus chemotherapy alone for treating advanced non-small cell lung cancer: a meta-analysis. PloS one 2015, 10: e0131278. Xiao W, Zeng X, Lin H, Han K, Jia H-Z, Zhang X-ZJCc: Dual stimuli-responsive multi-drug delivery system for the individually controlled release of anti-cancer drugs. Chemical communications 2015, 51: 1475-1478. Kibria G, Hatakeyama H, Harashima HJAopr: Cancer multidrug resistance: mechanisms involved and strategies for circumvention using a drug delivery system. Archives of pharmacal research 2014, 37: 4-15. Jang Y, Kim A, Moon JJ, Lee JY, Park HJAMT: Novel bioengineering strategies for drug delivery systems. Applied Materials Today 2023, 33: 101834. Maeda H, Wu J, Sawa T, Matsumura Y, Hori KJJocr: Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. Journal of controlled release 2000, 65: 271-284. Duncan RJNrc: Polymer conjugates as anticancer nanomedicines. Nature reviews cancer 2006, 6: 688-701. Kumari P, Ghosh B, Biswas SJJodt: Nanocarriers for cancer-targeted drug delivery. Journal of drug targeting 2016, 24: 179-191. Hossen S, Hossain MK, Basher M, Mia M, Rahman M, Uddin MJJJoar: Smart nanocarrier-based drug delivery systems for cancer therapy and toxicity studies: A review. Journal of advanced research 2019, 15: 1-18. Wilczewska AZ, Niemirowicz K, Markiewicz KH, Car HJPr: Nanoparticles as drug delivery systems. Pharmacological reports 2012, 64: 1020-1037. Xu W, Riikonen J, Lehto V-PJIjop: Mesoporous systems for poorly soluble drugs. International journal of pharmaceutics 2013, 453: 181-197. Lin Y-S, Abadeer N, Haynes CLJCC: Stability of small mesoporous silica nanoparticles in biological media. Chemical Communications 2011, 47: 532-534. Taylor KM, Kim JS, Rieter WJ, An H, Lin W, Lin WJJotACS: Mesoporous silica nanospheres as highly efficient MRI contrast agents. Journal of the American Chemical Society 2008, 130: 2154-2155. Lee CH, Cheng SH, Wang YJ, Chen YC, Chen NT, Souris J, Chen CT, Mou CY, Yang CS, Lo LWJAFM: Near ‐infrared mesoporous silica nanoparticles for optical imaging: characterization and in vivo biodistribution. Advanced Functional Materials 2009, 19: 215-222. Lu F, Wu SH, Hung Y, Mou CYJS: Size effect on cell uptake in well ‐suspended, uniform mesoporous silica nanoparticles. Small 2009, 5: 1408-1413. He Q, Zhang Z, Gao Y, Shi J, Li YJS: Intracellular localization and cytotoxicity of spherical mesoporous silica nano ‐and microparticles. Small 2009, 5: 2722-2729. He Q, Shi JJJoMC: Mesoporous silica nanoparticle based nano drug delivery systems: synthesis, controlled drug release and delivery, pharmacokinetics and biocompatibility. Journal of Materials Chemistry 2011, 21: 5845-5855. Meka AK, Jenkins LJ, Dàvalos-Salas M, Pujara N, Wong KY, Kumeria T, Mariadason JM, Popat AJP: Enhanced solubility, permeability and anticancer activity of vorinostat using tailored mesoporous silica nanoparticles. Pharmaceutics 2018, 10: 283. Bagwe RP, Hilliard LR, Tan WJL: Surface modification of silica nanoparticles to reduce aggregation and nonspecific binding. Langmuir 2006, 22: 4357-4362. Santra S, Liesenfeld B, Bertolino C, Dutta D, Cao Z, Tan W, Moudgil BM, Mericle RAJJoL: Fluorescence lifetime measurements to determine the core–shell nanostructure of FITC-doped silica nanoparticles: An optical approach to evaluate nanoparticle photostability. Journal of Luminescence 2006, 117: 75-82. Zhang Q, Wang J, Zhang H, Liu D, Ming L, Liu L, Dong Y, Jian B, Cai DJRa: The anticancer efficacy of paclitaxel liposomes modified with low-toxicity hydrophobic cell-penetrating peptides in breast cancer: an in vitro and in vivo evaluation. RSC advances 2018, 8: 24084-24093. Deshpande PP, Biswas S, Torchilin VPJN: Current trends in the use of liposomes for tumor targeting. Nanomedicine 2013, 8: 1509-1528. Derycke AS, Kamuhabwa A, Gijsens A, Roskams T, De Vos D, Kasran A, Huwyler J, Missiaen L, de Witte PAJJotNCI: Transferrin-conjugated liposome targeting of photosensitizer AlPcS 4 to rat bladder carcinoma cells. Journal of the National Cancer Institute 2004, 96: 1620-1630. Noble GT, Stefanick JF, Ashley JD, Kiziltepe T, Bilgicer BJTib: Ligand-targeted liposome design: challenges and fundamental considerations. Trends in biotechnology 2014, 32: 32-45. Xu J, Long Y, Ni L, Yuan X, Yu N, Wu R, Tao J, Zhang YJBc: Anticancer effect of berberine based on experimental animal models of various cancers: a systematic review and meta-analysis. BMC cancer 2019, 19: 1-20. Liu H, Huang C, Wu L, Wen BJO, therapy: Effect of evodiamine and berberine on miR-429 as an oncogene in human colorectal cancer. OncoTargets and therapy 2016 : 4121-4127. Kim S, Lee SY, Cho H-JJB, communications br: Berberine and zinc oxide-based nanoparticles for the chemo-photothermal therapy of lung adenocarcinoma. Biochemical and biophysical research communications 2018, 501: 765-770. Tian Y, Zhao L, Wang Y, Zhang H, Xu D, Zhao X, Li Y, Li JJAjoa: Berberine inhibits androgen synthesis by interaction with aldo-keto reductase 1C3 in 22Rv1 prostate cancer cells. Asian journal of andrology 2016, 18: 607. Wu K, Yang Q, Mu Y, Zhou L, Liu Y, Zhou Q, He BJIjoo: Berberine inhibits the proliferation of colon cancer cells by inactivating Wnt/β-catenin signaling. International journal of oncology 2012, 41: 292-298. Su Y-H, Tang W-C, Cheng Y-W, Sia P, Huang C-C, Lee Y-C, Jiang H-Y, Wu M-H, Lai I-L, Lee J-WJBeBA-MCR: Targeting of multiple oncogenic signaling pathways by Hsp90 inhibitor alone or in combination with berberine for treatment of colorectal cancer. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 2015, 1853: 2261-2272. Almatroodi SA, Alsahli MA, Rahmani AHJM: Berberine: An important emphasis on its anticancer effects through modulation of various cell signaling pathways. Molecules 2022, 27: 5889. Jiang X, Jiang Z, Jiang M, Sun Y: Berberine as a potential agent for the treatment of colorectal cancer. Frontiers in Medicine 2022, 9: 886996. Ge X, Lee Y-T, Yeh S-J: MD-Syn: Synergistic drug combination prediction based on the multidimensional feature fusion method and attention mechanisms. arXiv preprint arXiv:250107884 2025. Smitha K, Anitha A, Furuike T, Tamura H, Nair SV, Jayakumar RJC, Biointerfaces SB: In vitro evaluation of paclitaxel loaded amorphous chitin nanoparticles for colon cancer drug delivery. Colloids and Surfaces B: Biointerfaces 2013, 104: 245-253. Abu Samaan TM, Samec M, Liskova A, Kubatka P, Büsselberg DJB: Paclitaxel’s mechanistic and clinical effects on breast cancer. Biomolecules 2019, 9: 789. Armstrong DK, Bundy B, Wenzel L, Huang HQ, Baergen R, Lele S, Copeland LJ, Walker JL, Burger RAJNEJoM: Intraperitoneal cisplatin and paclitaxel in ovarian cancer. New England Journal of Medicine 2006, 354: 34-43. Jiménez-López J, El-Hammadi MM, Ortiz R, Cayero-Otero MD, Cabeza L, Perazzoli G, Martin-Banderas L, Baeyens JM, Prados J, Melguizo CJPR: A novel nanoformulation of PLGA with high non-ionic surfactant content improves in vitro and in vivo PTX activity against lung cancer. Pharmacological Research 2019, 141: 451-465. Sun J, Jiang L, Lin Y, Gerhard EM, Jiang X, Li L, Yang J, Gu ZJIjon: Enhanced anticancer efficacy of paclitaxel through multistage tumor-targeting liposomes modified with RGD and KLA peptides. International journal of nanomedicine 2017 : 1517-1537. Ma P, Chen J, Bi X, Li Z, Gao X, Li H, Zhu H, Huang Y, Qi J, Zhang YJAam, interfaces: Overcoming multidrug resistance through the GLUT1-mediated and enzyme-triggered mitochondrial targeting conjugate with redox-sensitive paclitaxel release. ACS applied materials & interfaces 2018, 10: 12351-12363. Murray S, Briasoulis E, Linardou H, Bafaloukos D, Papadimitriou CJCtr: Taxane resistance in breast cancer: mechanisms, predictive biomarkers and circumvention strategies. Cancer treatment reviews 2012, 38: 890-903. Cheng Y, Ji YJJocr: Mitochondria-targeting nanomedicine self-assembled from GSH-responsive paclitaxel-ss-berberine conjugate for synergetic cancer treatment with enhanced cytotoxicity. Journal of controlled release 2020, 318: 38-49. Xu C, Zheng L, Li D, Chen G, Gu J, Chen J, Yao Q: CXCR4 overexpression is correlated with poor prognosis in colorectal cancer. Life sciences 2018, 208: 333-340. Kollmar O, Rupertus K, Scheuer C, Nickels RM, Haberl GC, Tilton B, Menger MD, Schilling MK: CXCR4 and CXCR7 regulate angiogenesis and CT26. WT tumor growth independent from SDF ‐1. International journal of cancer 2010, 126: 1302-1315. Kurcinski M, Jamroz M, Blaszczyk M, Kolinski A, Kmiecik S: CABS-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site. Nucleic acids research 2015, 43: W419-W424. Inc N: Neurosnap: An online platform for computational biology and chemistry. 2022. Brand S, Dambacher J, Beigel F, Olszak T, Diebold J, Otte J-M, Göke B, Eichhorst STJEcr: CXCR4 and CXCL12 are inversely expressed in colorectal cancer cells and modulate cancer cell migration, invasion and MMP-9 activation. Experimental cell research 2005, 310: 117-130. Shin H-N, Moon H-H, Ku J-LJIjomm: Stromal cell-derived factor-1α and macrophage migration-inhibitory factor induce metastatic behavior in CXCR4-expressing colon cancer cells. International journal of molecular medicine 2012, 30: 1537-1543. Egorova A, Kiselev A, Hakli M, Ruponen M, Baranov V, Urtti AJTJoGMAcdjfrotsogt, applications ic: Chemokine ‐derived peptides as carriers for gene delivery to CXCR4 expressing cells. The Journal of Gene Medicine: A cross ‐disciplinary journal for research on the science of gene transfer and its clinical applications 2009, 11: 772-781. Dubey R, Rajesh Y, More MJMTP: Synthesis and characterization of SiO2 nanoparticles via sol-gel method for industrial applications. Materials Today: Proceedings 2015, 2: 3575-3579. Jiang X, Tang X, Tang L, Zhang B, Mao HJCI: Synthesis and formation mechanism of amorphous silica particles via sol–gel process with tetraethylorthosilicate. Ceramics International 2019, 45: 7673-7680. Chaudhary Z, Khan GM, Abeer MM, Pujara N, Tse BW-C, McGuckin MA, Popat A, Kumeria TJBs: Efficient photoacoustic imaging using indocyanine green (ICG) loaded functionalized mesoporous silica nanoparticles. Biomaterials science 2019, 7: 5002-5015. Liu J, Jiang X, Ashley C, Brinker CJJJotACS: Electrostatically mediated liposome fusion and lipid exchange with a nanoparticle-supported bilayer for control of surface charge, drug containment, and delivery. Journal of the American Chemical Society 2009, 131: 7567-7569. Dong J, Wu Z, Xu H, Ouyang D: FormulationAI: a novel web-based platform for drug formulation design driven by artificial intelligence. Briefings in Bioinformatics 2023, 25 . Müller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN: Involvement of chemokine receptors in breast cancer metastasis. nature 2001, 410: 50-56. Wang Y, Wang Q, Wang X, Yao P, Dai Q, Qi X, Yang M, Zhang X, Huang R, Yang J, et al: Docetaxel-loaded pH/ROS dual-responsive nanoparticles with self-supplied ROS for inhibiting metastasis and enhancing immunotherapy of breast cancer. Journal of Nanobiotechnology 2023, 21: 286. Lu J, Liong M, Zink JI, Tamanoi FJs: Mesoporous silica nanoparticles as a delivery system for hydrophobic anticancer drugs. small 2007, 3: 1341-1346. Slowing I, Trewyn BG, Lin VS-YJJotACS: Effect of surface functionalization of MCM-41-type mesoporous silica nanoparticles on the endocytosis by human cancer cells. Journal of the American Chemical Society 2006, 128: 14792-14793. Huh S, Wiench JW, Yoo J-C, Pruski M, Lin VS-YJCom: Organic functionalization and morphology control of mesoporous silicas via a co-condensation synthesis method. Chemistry of materials 2003, 15: 4247-4256. Hansen CB, Kao GY, Moase EH, Zalipsky S, Allen TMJBeBA-B: Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures. 1995, 1239: 133-144. Xu Y, Meng HJAC, Nanomedicine,, Biotechnology: Paclitaxel-loaded stealth liposomes: development, characterization, pharmacokinetics, and biodistribution. Artificial Cells, Nanomedicine, and Biotechnology 2016, 44: 350-355. Ruttala HB, Ko YTJC, Biointerfaces SB: Liposomal co-delivery of curcumin and albumin/paclitaxel nanoparticle for enhanced synergistic antitumor efficacy. Colloids and Surfaces B: Biointerfaces 2015, 128: 419-426. Additional Declarations No competing interests reported. Supplementary Files TableofContents.docx Cite Share Download PDF Status: Published Journal Publication published 22 Nov, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 08 Sep, 2025 Reviews received at journal 04 Sep, 2025 Reviews received at journal 31 Aug, 2025 Reviewers agreed at journal 11 Aug, 2025 Reviewers agreed at journal 10 Aug, 2025 Reviewers invited by journal 09 Aug, 2025 Editor assigned by journal 06 Aug, 2025 Submission checks completed at journal 06 Aug, 2025 First submitted to journal 06 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7307122","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":499087803,"identity":"6915d961-269b-4d43-be7f-9565085d690a","order_by":0,"name":"Yeonwoo Jang","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Yeonwoo","middleName":"","lastName":"Jang","suffix":""},{"id":499087804,"identity":"1768fad3-0353-45b9-80c9-1285124f0097","order_by":1,"name":"Amal Babu","email":"","orcid":"","institution":"Chonnam National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Amal","middleName":"","lastName":"Babu","suffix":""},{"id":499087805,"identity":"36658353-bcf9-4dcb-84e0-856ab9d8243b","order_by":2,"name":"Sahil Chahal","email":"","orcid":"","institution":"Chonnam National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Sahil","middleName":"","lastName":"Chahal","suffix":""},{"id":499087806,"identity":"217129cc-bcd0-4873-b26f-1e8e2687bb90","order_by":3,"name":"Arathy Vasukutty","email":"","orcid":"","institution":"Chonnam National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Arathy","middleName":"","lastName":"Vasukutty","suffix":""},{"id":499087807,"identity":"51908d11-06fd-437c-b215-1cf66f8866fe","order_by":4,"name":"James J. Moon","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"J.","lastName":"Moon","suffix":""},{"id":499087808,"identity":"27af0aee-0972-40b7-a3d8-4ef0fef29f73","order_by":5,"name":"In-Kyu Park","email":"","orcid":"","institution":"Chonnam National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"In-Kyu","middleName":"","lastName":"Park","suffix":""},{"id":499087809,"identity":"978ca329-393f-469a-8ca2-8c528c76c465","order_by":6,"name":"Hansoo Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBACAyBmBmI5ZAHitBiTriWxgWgt5uw9xp8LKmzS+9vPGDD8qGEwNm8goMWy54yZ9IwzabkzzuQYMPYcYzCTOUDIYTdyzJh52w7nbmDIMWDgbWCwkSDkMIP7b4w/A7WkG/C/MWD8S5SWGzwG0kAtCQYSOQbMQFvMCGs5k1YmzXMmzXDGjWcFh2WOSRgT1nL88ObPPBU28vz9yRsfvqmxMZxBSAsDAwciJg4wMBC0AwTYHxCjahSMglEwCkYyAACpzDiqTWQOBAAAAABJRU5ErkJggg==","orcid":"","institution":"Chung-Ang University","correspondingAuthor":true,"prefix":"","firstName":"Hansoo","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2025-08-06 07:53:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7307122/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7307122/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-025-03850-9","type":"published","date":"2025-11-22T15:58:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89071621,"identity":"992a8a35-c437-4c7c-b502-579127df526a","added_by":"auto","created_at":"2025-08-14 11:17:29","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":605456,"visible":true,"origin":"","legend":"\u003cp\u003eAI-guided design and overall assembly of the CXCR4-targeted dual-drug core–shell nanocarrier, FMSN(BBR)-CXCR4BPL(PTX). (A) AI-assisted targeting motif selection. Flexible docking (CABS-dock) and AlphaFold-based interface scoring (NeuroSNAP-AI) identified a 9-mer fragment of SDF-1 (pSDF-1, green), hereafter referred to as the CXCR4-binding peptide (CXCR4BP), that binds the extracellular pocket of the CXCR4 receptor with high confidence (ipTM 0.96, low PAE/PDE). (B) Nanocarrier concept and fabrication. (1) Mesoporous silica nanoparticle (MSN) synthesis → (2) surface functionalization with phosphonate/FITC to yield FMSN → (3) BBR loading [FMSN(BBR)] → (4) thin-film hydration of DSPC : DOTAP : cholesterol : lipids containing CXCR4BP (CXCR4BP-lipid) and PTX → (5) electrostatic self-assembly, yielding the core–shell nanocarrier, FMSN(BBR)-CXCR4BPL(PTX). The CXCR4BP on the liposomal corona enables receptor-mediated uptake by colorectal-carcinoma cells, while BBR resides in the silica core and PTX in the lipid shell for synergistic chemotherapy after intravenous administration.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7307122/v1/5ce5675f03e61f35e3063ae7.jpeg"},{"id":89073783,"identity":"70ae0079-3087-43f0-aa2e-1944130b7434","added_by":"auto","created_at":"2025-08-14 11:41:30","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":319205,"visible":true,"origin":"","legend":"\u003cp\u003eAI-guided identification of the CXCR4-binding peptide (CXCR4BP), its chemical conjugation to lipid, and physicochemical validation of CXCR4BP-liposomes. (A) Design-to-lipid workflow. Left: CABS-dock / NeuroSNAP-AI docking of the 9-mer CXCR4BP (green) on the extracellular pocket of CXCR4 (grey). Right: sulfhydryl-maleimide coupling of the cysteine-terminated peptide (KPVSLSYRC) with Maleimide-PEG(2000)-DSPE to give CXCR4BP-PEG(2000)-DSPE (CXCR4BP-lipid). (B) AlphaFold-multimer interface accuracy map (ipTM pairs); values approaching 1.0 (yellow) denote highly reliable chain–chain contacts. (C) Predicted aligned-error (PAE) heat-map; low PAE (\u0026lt;10 Å, purple/blue) indicates well-defined spatial relationships across the complex. (D) Predicted distance error (PDE) distribution further confirming high model precision. (E) LC/MS chromatograms of free CXCR4BP (top), Maleimide-PEG(2000)-DSPE (middle) and the purified conjugate (bottom), demonstrating successful peptide–lipid linkage (absence of individual starting-material peaks). (F) Hydrodynamic diameter of maleimide-liposomes and CXCR4BP-liposomes determined by NTA shows no significant size change after peptide insertion. (G) Zeta potential shifts upon introduction of the cationic peptide confirms surface display of CXCR4BP. (H) Protein content of maleimide-liposomes and CXCR4BP-liposomes quantified by BCA assay, validating peptide incorporation. Data represent mean ± SD.; n=3 (F, G), n=5 (H); *\u003cem\u003ep\u0026lt;0.05, \u003c/em\u003eand\u003cem\u003e ****p\u0026lt;0.0001 \u003c/em\u003evs\u003cem\u003e.\u003c/em\u003eControl/Model. ns, not significant. N.D., non-detection.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7307122/v1/ff0459bbf1555d5ac13ec839.jpeg"},{"id":89072565,"identity":"5e4864f2-09a0-4e95-a5ac-f498a45f097b","added_by":"auto","created_at":"2025-08-14 11:25:30","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":254652,"visible":true,"origin":"","legend":"\u003cp\u003ePhysicochemical characterization of phosphonate/FITC-functionalized mesoporous silica nanoparticles (FMSNs). (A) X-ray diffraction pattern showing the broad halo at 2θ ≈ 22°, characteristic of amorphous silica. (B) Left: FE-TEM image revealing radially aligned mesopores. Centre and right: STEM-EDS elemental maps of Si (cyan) and P (magenta) demonstrating homogeneous phosphonate distribution. (C) EDS spectrum confirming the presence of Si and P peaks. (E) FTIR spectra of unmodified MSNs (black) and phosphonate-grafted FMSNs (blue). Inset: enlargement of the 1300–1100 cm\u003csup\u003e-1 \u003c/sup\u003eregion highlighting the new P = O band (~1230 cm⁻¹) validating successful THMP functionalization. (F) Fluorescence emission spectra of FMSN dispersions (λ\u003csub\u003eex\u003c/sub\u003e = 480 nm) at increasing concentrations (0.0625 to 1 mg/mL). All samples exhibit a peak at 535 nm, attributable to covalently bound FITC; inset photograph shows the green fluorescence of FMSN. (B) Scale bar = 50 nm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7307122/v1/8fe545e3ab51c1cb10a3520f.jpeg"},{"id":89072898,"identity":"ef487154-05d6-4b97-9b3f-ca8f50b6b7a8","added_by":"auto","created_at":"2025-08-14 11:33:30","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":566272,"visible":true,"origin":"","legend":"\u003cp\u003eVerification of liposome coating on FMSNs, and characterization of FMSN(BBR)-CXCR4BPL(PTX). (A) Protein quantification of FMSN and FMSN-CXCR4BPL using BCA assay. (B) FE-TEM and STEM-EDS mapping images of silica (Si), phosphorous (P), and carbon (C) of FMSN-CXCR4BPL. (C) FE-SEM images of FMSN (left) and FMSN-CXCR4BPL (right). (D) CLSM images showing co-localization of FMSN (green), DiI-labeled CXCR4BPL (DiI-CXCR4BP, red). (E) MD-Syn \u003cem\u003ein-silico\u003c/em\u003e synergy screen of BBR with 38 oncology drugs across six human colorectal-cancer cell lines (HCT116, HT29, LoVo, RKO, SW620, SW837). PTX (highlighted in red) is the compound exhibiting \u0026gt; 0.9999 in every model (mean = 0.999999852). (F) Size of FMSN, FMSN-CXCR4BPL, and CXCR4BPL by NTA. (G) Zeta potential of FMSN, FMSN-CXCR4BPL, and CXCR4BPL by DLS. (H)-(I) Stability testing of FMSN-CXCR4BPL for 7 days. (J) BBR and PTX encapsulation efficiencies in FMSN-CXCR4BPL. (K) Cumulative drug release profile over time. Data are mean ± SD.; n=4 (A), n=5 (E), n=3 (F-J); **\u003cem\u003ep \u0026lt; 0.01, \u003c/em\u003eand\u003cem\u003e ****p \u0026lt; 0.0001\u003c/em\u003e.\u003cem\u003e 0001 \u003c/em\u003evs. Control/Model. ns, not significant. (B) Scale bar = 100 nm. (C) Scale bar = 100 nm. (D) Scale bar = 10 μm.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7307122/v1/db9ebb7488853b095b0e62ad.jpeg"},{"id":89072900,"identity":"277fbfd2-016b-4c77-ac3d-7b28cdf5d440","added_by":"auto","created_at":"2025-08-14 11:33:30","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":702574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn-vitro\u003c/em\u003e biocompatibility of FMSN and FMSN-CXCR4BPL. (A)-(B) Live/Dead staining fluorescence microscopy images of CT26 and hASC cells after 48 h of inoculation with FMSN and FMSN-CXCR4BPL (1, 10, and 100 μg/mL). Live cells were stained with calcein AM (green), and dead cells were stained with ethidium homodimer (EthD-1) (red). (C)-(D) Cell viability assessed using the CCK-8 assay of CT26 and hASC cells after 48 h of inoculation with FMSN and FMSN-CXCR4BPL (1, 10, and 100 μg/mL). Data are mean ± SD.; n=3 (C, D); \u003cem\u003e**p \u0026lt; 0.01, \u003c/em\u003eand \u003cem\u003e***p \u0026lt; 0.001 \u003c/em\u003evs. Control/Model. ns, not significant\u003cem\u003e.\u003c/em\u003e (A) Scale bar = 50 μm. (B) Scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7307122/v1/0be4391c79aa9848f2700d6c.jpeg"},{"id":89072569,"identity":"b5e173fb-7ff8-45ce-96de-28bce801d33f","added_by":"auto","created_at":"2025-08-14 11:25:30","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":588187,"visible":true,"origin":"","legend":"\u003cp\u003eCXCR4-mediated cancer-targeting ability of FMSN-CXCR4BPL \u003cem\u003ein-vitro\u003c/em\u003e and \u003cem\u003ein-vivo\u003c/em\u003e. (A-B) CLSM images of CT26 cells with FMSN and FMSN-CXCR4BPL (green) for 0.5, 1, 3, and 5 h. The nucleus was stained with DAPI (blue) and the cytoplasm with Rhodamine B (red). (B) Percentage of nanocarrier-positive cells quantified from (A) using ImageJ. (C) and (D) Flow cytometry analysis of the intracellular localization of FMSN and FMSN-CXCR4BPL after 0.5, 1, 3, and 5 h of incubation. (E) Whole-body IVIS images of CT26-tumor-bearing mice at 2 , 4 , 6, 8, 24, and 48 h post-intravenous administration of FMSN and FMSN-CXCR4BPL. (F) \u003cem\u003eEx-vivo\u003c/em\u003e fluorescence images of excised tumors and major organs 48 h post-injection. (G) Quantitative radiant efficiency of excised tumor and major organs 48 h after injection.Data are presented as mean ± SD.; n=3 (D, G), n= 6 (B); \u003cem\u003e***p \u0026lt; 0.001, \u003c/em\u003eand \u003cem\u003e****p \u0026lt; 0.0001 \u003c/em\u003evs. Control/Model. ns, not significant. (A) Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7307122/v1/f46d0186866591f08c38e586.jpeg"},{"id":89071636,"identity":"c9300e7b-0c50-4719-9534-ae296dabaca4","added_by":"auto","created_at":"2025-08-14 11:17:30","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":848476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn-vitro\u003c/em\u003ecancer migration inhibition and cytotoxicity effects of FMSN(BBR)-CXCR4BPL(PTX). (A) Scratch-wound assay in CT26 cells monolayers treated with BBR (1 μg/mL)/ PTX (0.4 μg/mL), FMSN including BBR (1 μg/mL)/ PTX (0.4 μg/mL), and FMSN-CXCR4BPL including BBR (1 μg/mL)/ PTX (0.4 μg/mL). Phase-contrast images were taken at 0, 6, 12, and 24 h. Dashed lines mark the initial wound edges. (B) Quantification of relative gap area versus time. FMSN(BBR)-CXCR4BPL(PTX) suppresses lateral migration most effectively. (C) CCK-8 viability assay of CT26 cells after 24, 48, and 72 h exposure to the same formulations. Targeted core–shell nanoparticles reduce viability significantly more than the free-drug mixture or non-targeted particles. (D) Live/dead staining (calcein-AM, green; EthD-1, red) at 24, 48, and 72 h corroborates the quantitative viability data. Extensive red fluorescence is observed only in the FMSN(BBR)-CXCR4BPL(PTX) group at 72 h. Data are presented as mean ± SD.; n=4 (B, C); \u003cem\u003e*p \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**p \u0026lt; 0.01\u003c/em\u003e, \u003cem\u003e***p \u0026lt; 0.001\u003c/em\u003e, and \u003cem\u003e****p \u0026lt; 0.0001 \u003c/em\u003evs\u003cem\u003e.\u003c/em\u003e Control/Model. ns, not significant. (A) Scale bar = 20 μm. (D) Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7307122/v1/84c6b3fea58ab1eac452a80b.jpeg"},{"id":89071639,"identity":"59daa43b-e686-4651-b216-bbb25ee55e81","added_by":"auto","created_at":"2025-08-14 11:17:30","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":980140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn-vivo\u003c/em\u003e antitumor activity of FMSN(BBR)-CXCR4BPL(PTX) in a CT26 subcutaneous mouse model. BALB/c mice bearing ∼100 mm\u003csup\u003e3\u003c/sup\u003e CT26 tumor received a single tail-vein dose of PBS, free BBR, free PTX, free BBR/PTX combination, non-targeted FMSN(BBR/PTX) or targeted FMSN(BBR)-CXCR4BPL(PTX) (100 µL; BBR 5 mg/kg) on day 0. (A) Tumor growth curves over 21 days. (B) Endpoint tumor weights of mice. (C) Photographs of excised tumors. (D) Body weight evolution demonstrating systemic tolerance. (E) Spleen weights and (F) Representative spleen images showing reduced splenomegaly in the targeted group. (G) H\u0026amp;E staining of heart, liver, spleen, lung, and kidney. (H) Ki67 staining of tumor tissue sections to visualize tumor proliferation. (I) TUNEL staining to assess tumor apoptosis. (J) CD31 staining to analyze tumor angiogenesis. (K-M) Quantification of the mean fluorescence intensity of Ki67-positive cells, TUNEL-positive cells, and CD31 positive cells. Data presented are mean ± SEM.; n=3 (K, L, M), n=4 (A, B, D, E); \u003cem\u003e*p \u0026lt; 0.05, ***p \u0026lt; 0.001, \u003c/em\u003eand \u003cem\u003e****p \u0026lt; 0.0001 \u003c/em\u003evs. Control/Model. ns, not significant\u003cem\u003e.\u003c/em\u003e (G) Scale bar = 50\u0026nbsp;μm. (H-J) Scale bar = 20\u0026nbsp;μm.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7307122/v1/80f7c694b3cba6682cd971ed.jpeg"},{"id":96920085,"identity":"999f98a7-b77e-4b4e-8edc-36dfd70e6d4c","added_by":"auto","created_at":"2025-11-27 14:14:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8756374,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7307122/v1/33d79c14-5e77-4682-a194-cd3832481dca.pdf"},{"id":89071622,"identity":"ed01f664-73b0-4461-a23b-7ecabfb1d4e0","added_by":"auto","created_at":"2025-08-14 11:17:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":192683,"visible":true,"origin":"","legend":"","description":"","filename":"TableofContents.docx","url":"https://assets-eu.researchsquare.com/files/rs-7307122/v1/ab2a4136d181f847a8b7e3c8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"AI-guided design of a CXCR4-targeted core–shell nanocarrier for co-delivery of berberine/paclitaxel in cancer therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCombination chemotherapy, simultaneous administration two or more mechanistically distinct agents, has long been recognized as a s a means of improving clinical response and delaying chemoresistance in solid tumors. Typically, mono-chemotherapy, which relies on a single agent, is not sufficient to effectively inhibit tumor growth due to its one-dimensional action mechanism. This results in accelerating tumor relapse and the emergence of chemoresistance mutations.[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] In contrast, combination chemotherapy represents an attractive strategy for cancer treatment because the agents used have complementary mechanisms of action against cancer cells, which serve to strengthen and activate alternative pathways.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Additionally, combination chemotherapy not only achieves synergistically outcomes but also helps prevent the development of drug resistance in cancer cells.[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] In clinical and preclinical studies, combination chemotherapy has shown a more robust anti-cancer response and improved survival rates than mono-chemotherapy.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eNanoparticle-mediated drug delivery systems, such as inorganic nanoparticles, lipid nanoparticles, polymeric nanoparticles, and nano-scaffolds, are currently undergoing preclinical and clinical development for combination chemotherapy by delivering multiple drugs to various cancers, offering benefits over conventional anticancer therapy.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] Nanoparticle-mediated drug delivery systems show passive cancer-targeting ability through the enhanced permeability and retention (EPR) effects, as well as active targeting capability by introducing receptor-specific ligands on the carriers.[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] Additionally, nanoparticles enable sustainable drug release, prevent degradation of agents, and minimize adverse effects, ultimately enhancing therapeutic efficacy.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] Therefore, nanoparticle-mediated delivery of multiple therapeutic agents is an advanced approach due to the various physicochemical and pharmacological properties of nanoparticles for combination chemotherapy. Among various nanoparticle-mediated drug delivery systems, mesoporous silica nanoparticles (MSNs) are considered a promising delivery system for the development of cancer therapies. This is due to their many advantages, such as surface functionality, size tunability, high intrinsic stability, and high drug encapsulation efficiency resulting from their unique mesoporous structure.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] While numerous published papers have successfully demonstrated the therapeutic effect of MSNs, very few \u003cem\u003ein-vivo\u003c/em\u003e experiments have been found in the literature. This is primarily because of the low stability of MSNs in a biological medium, which leads to rapid accidental uptake by the reticuloendothelial system (RES).[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] The dispersity of MSNs has been difficult to maintain, often resulting in irreversible aggregation.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] Moreover, the cytotoxicity of nanoscale MSN is significantly high at concentrations\u0026thinsp;\u0026asymp;\u0026thinsp;25 \u0026micro;g/mL.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Additionally, MSNs lack specific cancer-targeting ability, which can lead to side effects.\u003c/p\u003e\u003cp\u003eTo address these critical issues, we employed two engineering strategies: (1) functionalization of MSNs through surface modification, and (2) coating functionalized MSNs (FMSNs) with liposomes. MSNs are surface-modified with various moieties (e.g., amino, phosphonate, and carboxylate groups) to prevent false positive/negative signals and maintain the MSNs well-dispersed in aqueous solution with minimal to no aggregation. The positively or negatively charged MSNs improve solubility in biological solutions through electrostatic interactions, resulting in high permeability.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] Additionally, MSNs can be covalently bound to fluorescent dye molecules (e.g., fluorescein isothiocyanate [FITC] molecules) for bioimaging purposes.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] The FMSNs can also be coated with liposomes that have opposite charges to enhance solubility and reduce cytotoxicity. Liposomes are a clinically proven delivery system as they exhibit non-immunogenicity, good biocompatibility, high delivery efficiency, and ability to encapsulate hydrophobic and hydrophilic drugs.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] Furthermore, ligand-conjugated liposomes possess active targeting abilities towards cancer.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] Therefore, liposome-coated FMSNs could be used as a promising multidrug delivery nanoplatform for cancer treatment.\u003c/p\u003e\u003cp\u003eThe selection of complementary drugs is crucial in combination chemotherapy, where distinct mechanisms maximize tumor eradication. Berberine (BBR) is an alkaloid with broad antitumor activity, including against colorectal cancer, mediated by inhibition of angiogenesis and proliferation through cell-cycle arrest, autophagy modulation, and apoptosis induction.[\u003cspan additionalcitationids=\"CR32 CR33 CR34\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] BBR preferentially accumulates in the mitochondria of cancer cells, where it dissipates the mitochondrial membrane potential, elevates reactive-oxygen species (ROS), and triggers mitochondrial permeability transition, culminating in apoptosis.[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] BBR further enforces AMP-activated protein kinase (AMPK)-driven metabolic reprogramming and blocks epithelial\u0026ndash;mesenchymal transition in colorectal cancer cells, thereby suppressing tumor growth and metastasis.[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] While BBR demonstrates notable antitumor efficacy with relatively low toxicity, its ability to induce complete remission remains limited, necessitating combination strategies to enhance therapeutic outcomes.\u003c/p\u003e\u003cp\u003e To rationally choose a co-therapeutic for BBR, we applied the AI-driven drug-synergy platform (MD-Syn) to screen FDA-approved oncology agents across multiple colorectal cancer cell models.[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] Paclitaxel (PTX) consistently emerged as the top synergistic candidate. PTX, a microtubule-stabilizing agent used in breast, ovarian, lung, and colorectal cancers,[\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] not only arrests mitotic but also perturbs mitochondrial function to amplify apoptosis in malignant cells.[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] Its clinical efficacy, however, is often limited by multidrug resistance (MDR).[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] Because BBR acts through mitochondrial and metabolic pathways distinct from microtubule stabilization, the BBR/PTX combination offers complementary cytotoxic mechanisms while mitigating MDR, a premise supported by AI-predicted synergy and explored experimentally here.[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eIn this study, we integrate (i) AI-guided ligand discovery and drug-synergy selection, (ii) AI-rational drug allocation (PTX in the lipid shell, BBR in the FMSN core), and (iii) liposome-coated silica engineering to create FMSN(BBR)-CXCR4BPL(PTX), which is a C-X-C chemokine receptor type 4 (CXCR4)-targeted core-shell nanocarrier loaded with BBR and PTX as a multidrug delivery system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). CXCR4 is overexpressed in colorectal carcinoma and correlates with metastasis and poor prognosis.[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] To confer CXCR4 selectivity we implemented a dual-AI peptide screen: CABS-dock and NeuroSNAP-AI [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. We demonstrate that the platform (i) displays high dual-drug encapsulation efficiency, (ii) releases both agents in a sustained fashion, (iii) remains non-toxic up to 100 \u0026micro;g/mL, (iv) accumulates selectively in CXCR4-positive tumors, and (v) produces synergistic anti-proliferative and anti-migratory effects \u003cem\u003ein-vitro\u003c/em\u003e and significant tumor regression \u003cem\u003ein-vivo\u003c/em\u003e without systemic toxicity. Collectively, these findings showcase how AI-assisted selection of synergistic drug pairs and rational nanocarrier design can accelerate the development of next-generation, receptor-targeted combination nanotherapies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"2. Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. AI-guided selection of the CXCR4-binding peptide and fabrication of CXCR4BP-liposomes\u003c/h2\u003e\u003cp\u003eAn AI-driven pipeline was first used to identify an optimal ligand for CXCR4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Flexible docking of SDF-1/CXCR4\u0026ndash;derived sequences with CABS-dock ranked a 9-mer fragment of SDF-1 (Lys\u0026ndash;Cys) highest.[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] Subsequent AlphaFold-multimer analysis with NeuroSNAP-AI returned an inter-protein TM-score (ipTM) of 0.95 and uniformly low predicted-aligned-error (PAE) values (\u0026lt;\u0026thinsp;10 \u0026Aring;) across the interface, while the predicted-distance-error (PDE) map displayed a tight blue\u0026ndash;purple distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These metrics indicate a high-confidence, well-defined binding pose; the peptide was therefore designated CXCR4-binding peptide (CXCR4BP). To anchor the ligand in a lipid membrane, CXCR4BP was conjugated to Maleimide-PEG(2000)-DSPE via a sulfhydryl\u0026ndash;maleimide reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, right). LC/MS confirmed disappearance of the free peptide and maleimide peaks, giving a single product at the expected m/z (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) with a coupling yield was 97.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2%.\u003c/p\u003e\u003cp\u003eCXCR4BP-liposomes (CXCR4BPLs) were then prepared in a single thin-film hydration method. DSPC, DOTAP, cholesterol and the freshly synthesized CXCR4BP-lipid (67 : 8 : 20 : 5 mol %) were co-dissolved to form CXCR4BPL. For the controls, maleimide-PEG(2000)-DSPE was inserted into liposomes without CXCR4BP. Compared with peptide-free control liposomes (Mal-liposome), CXCR4BP-liposomes whose mean diameter (122\u0026thinsp;\u0026plusmn;\u0026thinsp;4 nm) was indistinguishable from maleimide control liposomes (119\u0026thinsp;\u0026plusmn;\u0026thinsp;5 nm; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). In contrast, the ζ-potential shifted from 12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 mV to 26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 mV after peptide insertion, consistent with surface display of the mildly cationic ligand (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). A bicinchoninic-acid (BCA) assay quantified 112.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2 \u0026micro;g peptide per mg lipid, confirming successful and reproducible incorporation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003eCollectively, these results validate (i) the computationally selected CXCR4BP as a high-affinity ligand, (ii) its efficient chemical linkage to DSPE lipid, and (iii) the generation of size-conserved liposomes bearing a peptide-rich corona, laying the foundation for a subsequent core\u0026ndash;shell nanocarrier assembly.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Synthesis and physicochemical characterization of phosphonate/FITC-functionalized MSNs (FMSNs)\u003c/h2\u003e\u003cp\u003eMSNs were produced by a base-catalyzed sol\u0026ndash;gel reaction and subsequently co-functionalized with 3-trihydroxysilyl\u0026shy;propyl\u0026shy;methyl\u0026shy;phosphonate (THMP) and FITC-APTMS (see Methods). FITC offers bioimaging function, while phosphonate confers high solubility in aqueous solution and cationic liposome coating.\u003c/p\u003e\u003cp\u003eThe broad halo at 2θ\u0026thinsp;\u0026asymp;\u0026thinsp;22\u0026deg; in the X-ray diffractometer (XRD) profile confirms the amorphous silica framework is retained after surface modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] The field emission-transmission electron microscope (FE-TEM) reveals well-defined and radially aligned mesopores with an average particle diameter of ~\u0026thinsp;150 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cb\u003eleft\u003c/b\u003e). Scanning transmission electron microscopy with energy dispersive spectroscopy (STEM-EDS) elemental maps display that phosphorus (magenta) is homogeneously distributed throughout the silica matrix (cyan) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cb\u003eright\u003c/b\u003e), while the corresponding EDS spectrum exhibits clear Si and P peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), further confirming surface modification. The exact amount of phosphorus was identified as 6.52 wt% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results confirmed that the P element was successfully functionalized onto the MSNs. Fourier transform infrared spectrophotometer (FTIR) comparison of pristine MSNs and functionalized FMSNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) provides further evidence of successful phosphonate functionalization. In addition to the common Si-O-Si bands at 1080 and 810 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e,[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] the FMSN trace displays a new P\u0026thinsp;=\u0026thinsp;O stretching band at ~\u0026thinsp;1230 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (inset), whereas the parent MSN lacks these features, indicative of successful THMP attachment on FMSNs. FITC labelling endows the particles with bright green fluorescence, and the intensity of which increased linearly with dispersion concentration and exhibited a maximum at 535 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, inset photograph).\u003c/p\u003e\u003cp\u003eCollectively, these results demonstrate that the dual functionalization strategy preserves the ordered mesoporous architecture while imparting aqueous dispersibility, traceable fluorescence and a phosphonate-rich surface for subsequent liposome coating and drug loading.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSTEM-EDS elemental composition of phosphonate/FITC-functionalized mesoporous silica nanoparticles (FMSNs). Quantitative analysis confirms successful phosphonate grafting, with phosphorus accounting for 6.52 wt % (5.95 at %).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eElement\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLine Type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ek Factor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ek Factor type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAbsorption Correction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eWt%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eWt% Sigma\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAtomic%\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK series\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.807\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTheoretical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK series\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTheoretical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e93.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e94.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK series\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTheoretical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e5.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal:\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. CXCR4BPL coating on FMSN and characterization of FMSN-CXCR4BPL\u003c/h2\u003e\u003cp\u003eThe self-assembly was initiated by hydrating a thin film of CXCR4BPL with FMSN using probe-sonication to facilitate electrostatic interaction between the negatively charged FMSN and cationic CXCR4BPL.[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] BCA assay, FE-TEM with STEM-EDS mapping, field-emission scanning electron microscopy (FE-SEM), and Confocal laser-scanning microscopy (CLSM) were used to confirm the liposome coating.\u003c/p\u003e\u003cp\u003eFollowing the liposome coating, FMSN-CXCR4BPLs exhibited a significantly higher protein content than FMSNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), attributed to the peptide portion of CXCR4BPLs. The FE-TEM combined with STEM-EDS mapping visualizes a clear core\u0026ndash;shell architecture of FMSN-CXCRBPL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Silicon and phosphorus signals co-localize in the FMSN cores, whereas carbon is confined to the liposome shells within the same regions of the FMSN-CXCR4BPL particles. FE-SEM highlights the resulting surface transformation from a smooth silica texture to a blurred, uniformly coated surface indicative of the surrounding lipid layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In addition, CLSM shows co-localization of FITC-FMSN (green) and DiI-labelled CXCR4BP (red) signals, confirming that each FMSN core is uniformly enveloped by the lipid shell (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eWe further characterized FMSN, FMSN-CXCR4BPL, and CXCR4BPL. Dynamic-light-scattering (DLS) analysis indicates that the hydrodynamic diameter of FMSN (\u0026asymp;\u0026thinsp;130 nm) is maintained after coating (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), confirming that liposome coating does not appreciably change particle size. As expected, the ζ-potential shifts from \u0026minus;\u0026thinsp;7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 mV to +\u0026thinsp;8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 mV due to the positive charge of peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). The core\u0026ndash;shell nanocarrier is colloidally stable in PBS for at least 7 days, with no significant change in PDI (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) or average size (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSTEM-EDS elemental composition of liposome-coated nanoparticles (FMSN-CXCR4BPL). The marked increase in carbon (~\u0026thinsp;39 wt %) relative to bare FMSNs, together with the residual silicon and trace phosphorus from the core, confirms the presence of an organic lipid shell surrounding the silica core.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eElement\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLine Type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ek Factor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ek Factor type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAbsorption Correction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eWt%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eWt% Sigma\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAtomic%\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK series\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTheoretical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e39.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e51.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK series\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.455\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTheoretical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e35.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e34.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK series\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTheoretical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e24.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e13.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK series\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTheoretical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal:\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Synergy screening and BBR/PTX co-loading of FMSN-CXCR4BPL\u003c/h2\u003e\u003cp\u003eBBR was chosen as the anchor drug because it triggers cell-cycle arrest, AMP-activated protein kinase (AMPK)-driven metabolic reprogramming, and inhibition of epithelial\u0026ndash;mesenchymal transition in colorectal cancer cells, thereby suppressing tumor growth and metastasis.[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] While BBR exhibits broad antitumor activity and relatively low toxicity, its monotherapeutic potential remains limited due to incomplete tumor eradication and risk of recurrence, necessitating a rationally selected co-therapeutic agent to enhance overall efficacy. To identify a truly cooperative drug, we employed the MD-Syn AI platform to screen BBR against 38 FDA-approved oncology agents across six human colorectal-cancer cell lines (HCT116, HT29, LoVo, RKO, SW620, SW837),[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] which served as surrogates in lieu of a CT26 murine model. PTX emerged as the compound with a synergy-probability\u0026thinsp;\u0026gt;\u0026thinsp;0.9999 in every cell line (grand mean\u0026thinsp;=\u0026thinsp;0.999999852; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), justifying its selection for combination therapy.\u003c/p\u003e\u003cp\u003ePrior to formulation, we employed the open-source FormulationAI drug encapsulation module.[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] The model predicted encapsulation efficiencies of 52.56% (BBR in liposome) and 68.65% (PTX in liposome). According to these AI-guided results, we decided to encapsulate PTX in CXCR4BPL part and load BBR in FMSN. After one-pot self-assembly, the resulting core\u0026ndash;shell nanocarrier achieved BBR EE\u0026thinsp;=\u0026thinsp;78.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9% and PTX EE\u0026thinsp;=\u0026thinsp;75.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ), suggesting an optimized loading strategy for this core-shell loading combination. Additionally, the release kinetics of BBR and PTX from FMSN(BBR)-CXCR4BPL(PTX) demonstrated sustained release over a 72-h period in PBS (pH 7.4, 37\u0026deg;C), indicating prolonged drug availability that could enhance targeted cancer cells treatment while maintaining effective therapeutic levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). These results support the rationale for combining BBR with PTX, as the core\u0026ndash;shell architecture not only accommodates both drugs at high loading capacity but also ensures their sustained co-delivery, a crucial factor in achieving the predicted synergistic antitumor effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. \u003cem\u003eIn-vitro\u003c/em\u003e biocompatibility of FMSN-CXCR4BPL\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003ein-vitro\u003c/em\u003e biocompatibility of bare FMSN and lipid-coated FMSN-CXCR4BPL was assessed using a live/dead cell assay and CCK-8 assay on CT26 colon-carcinoma cells and human adipose-derived MSCs (hASC) at various concentrations after 48 h incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, CT26 and hASC exposed to bare FMSN at 100 \u0026micro;g/mL displayed an increase in dead cells (red fluorescent), whereas little or no red signal was detected at 1 \u0026micro;g/mL and 10 \u0026micro;g/mL. In contrast, cells treated with FMSN-CXCR4BPL retained an almost exclusively green fluorescence pattern even at 100 \u0026micro;g/mL, indicating minimal membrane damage. The lipid shell effectively masks the potential toxicity at high concentration of the FMSN cores (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These observations were corroborated by the CCK-8 assay results. For CT26, viability fell to 74.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5% with bare FMSN at 100 \u0026micro;g/mL but remained 93.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5% with FMSN-CXCR4BPL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). hASC showed a similar trend (bare FMSN 77.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8%; coated FMSN-CXCR4BPL 97.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5%, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). No significant difference was observed between the two formulations at 1 \u0026micro;g/mL or 10 \u0026micro;g/mL. These results agree with previous reports that uncoated MSNs become dose-limiting above \u0026asymp;\u0026thinsp;25 \u0026micro;g/mL owing to exposed silanol groups.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] The phospholipid shell passivates reactive silanols, thereby suppressing membrane disruption and improving serum compatibility. Because the CXCR4BP is present at only 0.5 mol % of total lipid, the shell itself is essentially non-toxic while conferring receptor-mediated targeting capability.\u003c/p\u003e\u003cp\u003eCollectively, the lipid corona transforms a dose-limited inorganic carrier into a biocompatible platform that remains safe at 100 \u0026micro;g/mL, which is a concentration exceeding typical therapeutic doses used in subsequent \u003cem\u003ein-vivo\u003c/em\u003e studies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. CXCR4-mediated cancer-targeting ability \u003cem\u003ein-vitro\u003c/em\u003e and \u003cem\u003ein-vivo\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo evaluate whether CXCR4BPs enhance tumor selectivity, we compared bare FMSN with FMSN-CXCR4BPL in CXCR4-overexpressing CT26 colon-carcinoma cells and in CT26-tumor-bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCLSM showed time-dependent internalization of both formulations, but FMSN-CXCR4BPL produced markedly stronger intracellular fluorescence from 3 h onward (green) than bare FMSN (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). ImageJ analysis confirmed that the percentage of nanoparticle-positive CT26 cells rose to 18.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% for FMSN-CXCR4BPL versus 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1% for FMSN after 3 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Flow cytometry analysis was conducted on CT26 cells to substantiate these findings with additional quantitative support. Flow cytometry histograms echoed this trend. FITC-positive populations appeared as early as 0.5 h and reached a three-fold higher mean fluorescence intensity for FMSN-CXCR4BPL at 3 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These data demonstrate efficient CXCR4-mediated attachment and internalization of FMSN-CXCR4BPL \u003cem\u003ein-vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eTo evaluation of CXCR4-mediated tumor-targeting ability of FMSN-CXCR4BP, IR780-loaded FMSNs and FMSN-CXCR4BPLs were injected i.v. (0.5 mg/kg IR780) to CT26-tumor-bearing BALB/c mice. Whole-body imaging revealed progressive tumor-specific fluorescence, peaking at 24\u0026ndash;48 h. Signal in the FMSN-CXCR4BPL group was consistently higher than in the FMSN group, indicating improved targeting and/or retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). \u003cem\u003eEx-vivo\u003c/em\u003e fluorescence imaging 48 h post-injection showed the strongest fluorescence in tumor relative to other organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Quantification confirmed a three-fold higher radiant efficiency in tumors treated with FMSN-CXCR4BPL compared with bare FMSN (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eTogether, the CLSM/flow cytometry \u003cem\u003ein-vitro\u003c/em\u003e data and the \u003cem\u003ein-vivo\u003c/em\u003e imaging results establish that the CXCR4-binding lipid corona confers robust and selective targeting to CXCR4-positive cancer cells, leading to enhanced tumor accumulation without off-target organ uptake.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.7.\u003c/b\u003e \u003cb\u003eIn-vitro\u003c/b\u003e \u003cb\u003eanticancer efficacy of FMSN(BBR)-CXCR4BPL(PTX)\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe targeted core\u0026ndash;shell nanocarrier (FMSN(BBR)-CXCR4BPL(PTX)) was compared with a free BBR/PTX mixture and a non-targeted silica formulation, FMSN(BBR/PTX).\u003c/p\u003e\u003cp\u003eBecause CXCR4 signaling is a key driver of tumor dissemination, we first assessed cell migration, a prerequisite for metastasis, alongside direct cytotoxicity.[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] In a scratch-wound assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), control monolayers almost completely closed\u0026thinsp;\u0026gt;\u0026thinsp;97% of the gap within 24 h, and free dual-drug mixture (BBR/PTX) reduced closure only modestly. Non-targeted FMSN(BBR/PTX) slowed migration further, yet a continuous front was still visible. In contrast, FMSN(BBR)-CXCR4BPL(PTX) preserved 93.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9% of the initial gap after 24 h, significantly higher than all other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eMetabolic activity measured by CCK-8 assay followed the same drug-dose\u0026ndash;dependent trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). After 72 h, cancer cell viability fell to 36.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9% with the targeted nanocarrier, whereas free drugs and non-targeted particles left 81.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7% and 68.9\u0026thinsp;\u0026plusmn;\u0026thinsp;20.1% viable cancer cells, respectively. Live/dead staining corroborated these data (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Extensive EthD-1 (red) fluorescence was evident only in the FMSN(BBR)-CXCR4BPL(PTX) group at 72 h.\u003c/p\u003e\u003cp\u003eThus, by combining CXCR4-specific delivery with complementary mitochondrial (BBR) and mitotic (PTX) mechanisms, the core\u0026ndash;shell nanocarrier not only kills CT26 cancer cells more efficiently but also blocks the migratory behavior that underlies metastasis, highlighting its dual anti-cancer potential.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. \u003cem\u003eIn-vivo\u003c/em\u003e antitumor efficacy of FMSN(BBR)-CXCR4BPL(PTX)\u003c/h2\u003e\u003cp\u003eBuilding on promising \u003cem\u003ein-vitro\u003c/em\u003e cytotoxicity and significant tumor accumulation, we evaluated the antitumor efficacy of FSMN(BBR/PTX) and FMSN(BBR)-CXCR4BPL(PTX) nanoparticles in a CT26 tumor-bearing mice model. When tumors reached approximately 100 mm\u003csup\u003e3\u003c/sup\u003e in volume, mice were randomly assigned to six treatment groups: PBS (control), free BBR, free PTX, a free BBR/PTX combination, FSMN(BBR/PTX), and FMSN(BBR)-CXCR4BPL(PTX).\u003c/p\u003e\u003cp\u003eA single i.v. administration of the CXCR4-targeted core\u0026ndash;shell nanocarrier produced the most pronounced tumor control among the six treatment arms. Tumors in the PBS control group expanded steadily, reaching\u0026thinsp;\u0026asymp;\u0026thinsp;1500 mm\u0026sup3; by day 21, whereas growth was only modestly delayed by free BBR or PTX alone. A combined injection of the two free drugs and the non-targeted FMSN(BBR/PTX) did slow tumor growth, yet a clear expansion persisted. In contrast, FMSN(BBR)-CXCR4BPL(PTX) virtually halted tumor enlargement, limiting the final volume to 14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8% of the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Tumor growth data were corroborated by endpoint tumor weights. the targeted nanocarrier reduced mean tumor mass from 4.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 g in PBS controls to 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 g, i.e. \u0026asymp; 13.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2% of the PBS control value (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). This weight reduction parallels the volumetric inhibition, and the excised tumors displayed the same hierarchy of mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). The results highlight the superior \u003cem\u003ein-vivo\u003c/em\u003e potency of FMSN(BBR)-CXCR4BPL(PTX) over free drugs or the non-targeted formulation.\u003c/p\u003e\u003cp\u003eSystemic tolerance was excellent, since no significant body-weight changes were observed in any group over 21 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), and H\u0026amp;E examination revealed no histopathological damage to heart, liver, spleen, lung, or kidney (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). Spleen weight served as an additional systemic read-out. In this model, tumor-driven inflammation produces pronounced splenomegaly.[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] Notably, untreated mice exhibited marked splenomegaly (876.2\u0026thinsp;\u0026plusmn;\u0026thinsp;68.9 mg), whereas the targeted formulation normalized spleen weight to 214.3\u0026thinsp;\u0026plusmn;\u0026thinsp;55.1 mg, roughly a four-fold reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). The reversal of splenomegaly therefore indicates that the targeted therapy not only shrinks the primary tumor but also alleviates tumor-induced systemic immune dysregulation, further underscoring its favorable safety\u0026ndash;efficacy profile.\u003c/p\u003e\u003cp\u003eTo further confirm the enhanced antitumor activity of FMSN(BBR)-CXCR4BPL(PTX), particularly in relation to cell proliferation, immunohistochemistry staining was performed. Ki-67 fluorescence fell to 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2% of control, indicating strong proliferation arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK). A TUNEL assay revealed a 6-fold rise in apoptotic nuclei relative to PBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL), while CD31 labelling showed a one-tenth drop in micro-vessel density, confirming potent anti-angiogenic action (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eM).\u003c/p\u003e\u003cp\u003eTogether, these results demonstrate that CXCR4-directed co-delivery of BBR and PTX achieves multi-modal tumor suppression, curbing proliferation, inducing apoptosis, and starving neo-vasculature without systemic toxicity, thereby validating the therapeutic promise of the FMSN(BBR)-CXCR4BPL(PTX) nanocarrier \u003cem\u003ein-vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eBy chaining three orthogonal AI modules, we established a rapid, data-driven route to a potent, receptor-targeted combination chemotherapy for colorectal cancer. (i) MD-Syn screening of 38 FDA-approved oncology drugs in six human colorectal-cancer cell lines (HCT116, HT29, LoVo, RKO, SW620, SW837) identified PTX as the compound with a\u0026thinsp;\u0026gt;\u0026thinsp;0.9999 probability of synergy with BBR, nominating the PTX/BBR pair for formulation. (ii) A dual-AI peptide pipeline, flexible docking with CABS-dock followed by AlphaFold-multimer scoring in NeuroSNAP-AI, pinpointed a high-affinity 9-mer SDF-1 fragment (CXCR4BP; ipTM\u0026thinsp;=\u0026thinsp;0.95) for selective targeting of the overexpressed CXCR4 receptor in colorectal tumors. (iii) FormulationAI predicted that loading BBR into FMSN cores and PTX into a CXCR4BPL shells. One-pot self-assembly yielded FMSN(BBR)-CXCR4BPL(PTX) with experimental entrapment efficiencies of 78.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9% (BBR) and 75.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4% (PTX), together with sustained co-release over 72 h. The ~\u0026thinsp;130 nm core\u0026ndash;shell nanocarrier curtailed scratch-wound closure by \u0026asymp;\u0026thinsp;94% and reduced metabolic viability to 36% in CXCR4-positive CT26 cells, vastly outperforming free drugs and non-targeted controls. In CT26-bearing mice, a single intravenous dose (5 mg/kg BBR equivalent) limited tumor volume and mass to ~\u0026thinsp;15% and ~\u0026thinsp;13% of PBS controls, normalized splenomegaly four-fold, and triggered strong anti-proliferative (\u0026darr;Ki-67), pro-apoptotic (\u0026uarr;TUNEL), and anti-angiogenic (\u0026darr;CD31) responses without body-weight loss or histopathological injury.\u003c/p\u003e\u003cp\u003eCollectively, these results demonstrate that AI-guided synergy scouting, ligand discovery, and drug-placement optimization, combined with modular nanocarrier engineering, can deliver high-payload, receptor-targeted combination therapeutics that couple potent efficacy with systemic tolerance. The workflow is readily transferable to other ligand\u0026ndash;receptor axes and drug pairs, offering a general blueprint for accelerating next-generation anticancer nanomedicines.\u003c/p\u003e"},{"header":"4. Experimental Section","content":"\u003cp\u003e\u003cem\u003eCell culture\u003c/em\u003e\u003c/p\u003e\u003cp\u003eColon carcinoma cells (CT26) and human adipose-derived stem cells (hASCs) were cultured in Dulbecco\u0026rsquo;s modified eagle\u0026rsquo;s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. Cell lines were incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePreparation of phosphonate/fluorescence-functionalized MSNs (FMSNs) and loading of BBR\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe FMSNs were synthesized using a base-catalyzed sol-gel method at high temperatures, modifying previous protocols.[\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] The FITC-labeled MSNs were prepared by reacting 1.4 mmol of FITC with 1.2 mmol of (3-aminopropyl)trimethoxy silane (APTMS) in ethanol under an argon atmosphere for 2 h. The FITC-APTMS product was then added to a co-condensation reaction of 5.0 mL of tetraethyl orthosilicate (TEOS), 1.0 g of hexadecyltrimethylammonium bromide (CTAB) in a mixture of 480 mL of distilled water, and 3.5 mL of 2 М sodium hydroxide. After 15 min of stirring at 80 ℃, 1.3 mL of 3-trihydroxysilylpropylmethylphosphonate (THMP) was added dropwise to the mixture. The reaction mixture was vigorously stirred at 80 ℃ for 2 h. Once the reaction was complete, the solution was cooled to ambient temperature, and the FITC-labeled nanoparticles were filtered and washed thoroughly with methanol using a fritted funnel under vacuum for 24 h. To remove the CTAB surfactants, 1.0 g of the nanoparticles were dissolved in a mixture of 100 mL of methanol and 1.0 mL of 37% hydrochloric acid. After refluxing the solution for 24 h, the nanoparticles were filtered and washed thoroughly with methanol through a fritted funnel under vacuum for 24 h. FMSN characterization was performed using an X-ray diffractometer (XRD). The validation of fluorescence of FMSN was determined using a microplate reader (SynergyTM H1, BioTek Instruments Inc.) by exciting the samples at 480 nm and measuring the emission spectra between 500 and 700 nm. Phosphonate-surface modification was verified using a Fourier transform infrared spectrophotometer (FTIR) and a field emission-transmission electron microscope (FE-TEM) coupled with energy-dispersive X-ray spectroscopy (EDS).\u003c/p\u003e\u003cp\u003eTo load drug molecules into the pores of the particles, the FMSNs were soaked in a concentrated solution containing the drugs. Typically, 100 mg of FMSNs were gently stirred in a solution containing 100 mg of BBR and ethanol: distilled water (7:3 v/v) solution for 48 h. After stirring, the mixture was centrifuged at 23,000 g for 10 min several times, and the supernatant was removed. The FMSN(BBR)s were then lyophilized.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePreparation and characterization of peptide-conjugated lipids\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePeptides that bind to CXCR4 and contain an additional cysteine at the N-terminus was conjugated with Maleimide-PEG(2000)-DSPE through a sulfhydryl-maleimide coupling reaction.[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] In brief, Maleimide-PEG(2000)-DSPE was first diluted in chloroform and then rotary evaporated at 37 ℃ and 120 rpm to create a thin film, which was then hydrated with pure water at 37\u0026deg;C. The peptides were dissolved in 0.1 М phosphate-buffered solutions (PBS) and mixed with Maleimide-PEG(2000)-DSPE (peptides/Maleimide-PEG(2000)-DSPE molar ratio of 1.2:1) and left to react overnight under argon gas at room temperature. The resulting mixture was then placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa for 48 h to remove the residual free peptides. After dialysis, the solution was dried through lyophilization. The peptide conjugation was analyzed using liquid chromatography-mass spectrometry (LC/MS) (Shimadzu, Japan) and bicinchoninic-acid (BCA) assay.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePreparation of FMSN(BBR)-CXCR4BPL(PTX)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe conventional thin-film hydration method was applied to prepare PTX-loaded liposomes[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] composed of 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane, cholesterol, and CXCR4BP-conjugated lipids (CXCR4BP-lipids) (67:8:20:5, molar ratio). Briefly, the lipids (lipid-to-FMSN mass ratio 10:1) and PTX (5%, wt/wt) were dissolved in chloroform, and the organic solvent was evaporated under reduced pressure at 70 ℃ using a hot drying oven for 2 h to form a thin film. The thin film was then hydrated with distilled water at 60 ℃ for 10 min. Following hydration, the lipid mixture was mixed with FMSN(BBR) solution and subjected to be stirred for 24 h. The mixture was then sonicated in an ice-water bath using probe sonication (on 2 s/off 2 s, 20 min, 26% amplitude). To remove any unencapsulated drugs, the FMSN(BBR)-CXCR4BPL(PTX) were centrifuged at 23,000 g for 10 min three times. Finally, the nanocarriers were dried through lyophilization. DiI-labeled CXCR4BPLs were prepared using the same methods, except that DiI was added before formation of the thin film.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePhysicochemical characterization of FMSN-CXCR4BPL\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe mean particle sizes and nanoparticle number concentration of FMSN, FMSN-CXCR4BPL, and CXCR4BPL were measured using nanoparticle tracking analysis (NTA) with a NanoSight NS300 (Malvern Instruments Ltd., Worcestershire, UK, camera level\u0026thinsp;=\u0026thinsp;12, detection threshold\u0026thinsp;=\u0026thinsp;5). The polydispersity indexes (PDIs) and zeta potentials (surface charges) of FMSN, FMSN-CXCR4BPL, and CXCR4BPL were determined via dynamic light scattering (DLS) using a Zetasizer Nano ZS system (Malvern Instruments Ltd., Worcestershire, UK). The stability of the core-shell nanocarriers was assessed by monitoring changes in size and PDI over 7 d. Morphology and composition were confirmed through FE-TEM coupled with EDS and field-emission scanning electron microscopy (FE-SEM). Additionally, CLSM was used to validate liposome coating and drug loading. Protein quantification of FMSN and FMSN-CXCR4BPL was performed using the bicinchoninic acid (BCA) assay.\u003c/p\u003e\u003cp\u003e\u003cem\u003eBBR or PTX encapsulation efficiency and release profiles\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe amount of loaded BBR or PTX was calculated using a microplate reader and high-performance liquid chromatography (HPLC, Agilent, CA, USA). To determine the amount of BBR or PTX in the core-shell nanocarrier, the FMSN-CXCR4BPL was processed with 1N HCl using a bath sonicator (100 W). Encapsulation efficiency (EE) was then calculated using the following equation:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:Drug\\:encapsulation\\:\\left(\\%\\right)=\\left(\\:\\frac{Amount\\:of\\:\\:drug\\:encapsulated\\:in\\:FMSN-CXCR4BPL}{Total\\:amount\\:of\\:drug\\:}\\right)\\times\\:100\\:$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTo measure the release profile of BBR or PTX, 400 \u0026micro;L of FMSN(BBR)-CXCR4BPL(PTX) was dialyzed against 14 mL of PBS (pH 7.4) with constant shaking (100 rpm) at 37\u0026deg;C. At different time intervals, 1 mL of the dialysis buffer solution was aliquoted for measurement and then replaced with an equal volume of fresh medium. The amount of released BBR or PTX was determined using a microplate reader or HPLC to quantify the concentration of each drug.\u003c/p\u003e\u003cp\u003e\u003cem\u003eBiocompatibility of FMSN-CXCR4BPL\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe LIVE/DEAD\u0026trade; Viability/Cytotoxicity Kit (Invitrogen\u0026trade;, USA) was used to assess the cytotoxicity of FMSN and FMSN-CXCR4BPL via fluorescence microscopy. CT26 and hASC cells were seeded at a density of 3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL in 24-well plates. After 24 h, the cells were exposed to FMSN or FMSN-CXCR4BPL (1, 10, and 100 \u0026micro;g/mL) in the cell-culture medium for 48 h. Following the 48 h incubation, the cells were treated with a live/dead solution (5 \u0026micro;L of calcein AM for live cells and 20 \u0026micro;L of ethidium homodimer (EthD-1) for dead cells in 10 mL of cell medium) for 30 min before observation under fluorescence microscopy. To evaluate the biocompatibility of FMSN and FMSN-CXCR4BPL, a cell counting kit-8 (CCK-8, DOJINDO, Japan) assay was performed. CT26 and hASC cells were seeded at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL in 96-well plates and incubated for 24 h. The cells were then exposed to FMSN or FMSN-CXCR4BPL (1, 10, and 100 \u0026micro;g/mL) for 48 h. After the 48 h incubation, the cell-culture medium was replaced with a 10% CCK-8 solution in each well and incubated at 37\u0026deg;C for an additional 2 h. The absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated based on the relative absorbance compared to the control absorbance. The use of hASCs was approved by the Institutional Review Board of Chung-Ang University Hospital (Approval No. 2151-005-463) and conducted in compliance with the principles outlined in the Declaration of Helsinki.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn-vitro cellular uptake of FMSN-CXCR4BPL for validation of cancer-cell-targeting ability via confocal laser-scanning microscopy (CLSM) and flow cytometry\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe cancer cell targeting ability was determined via CLSM. CT26 cells were seeded (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/chamber) on 4-well glass-chamber slides (SPL, Cell Culture Slide) in cell-culture medium. After 24 h, the cells were treated with fresh medium containing either 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e FMSN or 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e FMSN-CXCR4BPL for 0.5, 1, 3, and 5 h at 37\u0026deg;C. Following incubation, the chambers were washed at least three times with PBS (pH 7.4). The cells were then incubated in PBS with rhodamine phalloidin for 2 h for cytoplasm staining and DAPI for 3 min for nuclear staining. After washing several times with PBS, the chambers were mounted and examined using a Confocal Zeiss LSM 900 microscope, with ZEISS ZEN lite software used for setup. Flow cytometry was used to determine the cancer cell targeting ability. CT26 cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL) were cultured overnight at 37\u0026deg;C under a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The cellular uptake of FMSN and FMSN-CXCR4BPL was measured by incubating the cells with 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e FMSN and FMSN-CXCR4BPL in cell-culture medium for 0.5, 1, 3, and 5 h. Following incubation, the cells were washed twice with PBS, and the fluorescence was analyzed using a BD Accuri C6 Plus flow cytometer (BD Bioscience, USA). Cells cultured in the absence of the core-shell nanocarrier were used as the control. The experiments were repeated at least three times.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn-vitro cancer migration and cytotoxicity test of FMSN(BBR)-CXCR4BPL(PTX) for validation of anti-cancer effect.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eCell migration was assessed using a scratch assay. CT26 colon carcinoma cells were seeded at 8 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL in a 60-mm dish and incubated at 37\u0026deg;C for 24 h to form a confluent monolayer. Subsequently, scratches were made using a 10 \u0026micro;L micropipette tip. The scratched cell monolayers were then treated with cell-culture medium including BBR (1 \u0026micro;g/mL)/ PTX (0.4 \u0026micro;g/mL), FMSN containing BBR (1 \u0026micro;g/mL)/ PTX (0.4 \u0026micro;g/mL), and FMSN-CXCR4BPL containing BBR (1 \u0026micro;g/mL)/ PTX (0.4 \u0026micro;g/mL). After 0, 6, 12, and 24 h cell culture, images of the scratch areas were captured using phase-contrast microscopy and analyzed using Image J software. The extent of cell migration was determined using the following equation:\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:Relative\\:gap\\:area=\\left(\\:\\frac{gap\\:area\\:at\\:the\\:indicated\\:time\\:point}{gap\\:area\\:at\\:0\\:h\\:}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eQuantitative cytotoxicity was determined using a CCK-8 assay in 96-well plates. CT26 cells were seeded at a concentration of 7 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/mL and incubated for 24 h. The cells were then treated with BBR (1 \u0026micro;g/mL)/ PTX (0.4 \u0026micro;g/mL), FMSN containing BBR (1 \u0026micro;g/mL)/ PTX (0.4 \u0026micro;g/mL), and FMSN-CXCR4BPL containing BBR (1 \u0026micro;g/mL)/ PTX (0.4 \u0026micro;g/mL) for 24, 48, and 72 h. At each time point, 10% CCK-8 solution was added to each well and incubated at 37\u0026deg;C for an additional 2 h. The absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated as the relative absorbance compared to the control absorbance. Qualitative cytotoxicity of each group was assessed using the LIVE/DEAD\u0026trade; Viability/Cytotoxicity Kit (Invitrogen\u0026trade;, USA). CT26 cells at a density of 3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL were seeded in 24-well plates. After 24 h of culture, the cells were treated with BBR (1 \u0026micro;g/mL)/ PTX (0.4 \u0026micro;g/mL), FMSN containing BBR (1 \u0026micro;g/mL)/ PTX (0.4 \u0026micro;g/mL), and FMSN-CXCR4BPL containing BBR (1 \u0026micro;g/mL)/ PTX (0.4 \u0026micro;g/mL) for 24, 48, and 72 h. At each time point, the CT26 cells were incubated with the live/dead solution for 30 min before fluorescence microscopy.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn-vivo evaluation of tumor targetability and biodistribution\u003c/em\u003e\u003c/p\u003e\u003cp\u003eBalb/c mice were subcutaneously (s.c.) injected with 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CT26 cells into the right flank to induce tumor formation. Once the tumors reached an approximate volume of 100 mm\u0026sup3;, the mice received intravenous (i.v.) injections of FSMN and FMSN-CXCR4BPL loaded with IR780 for imaging (IR780 dose: 0.5 mg/kg). The biodistribution and tumor-targeting efficiency of FMSN-CXCR4BPL were monitored over time using optical imaging, with the assistance of the fluorescence-labeled organism bio-imaging system (FOBI; Neo-Science, Gyeonggi, Korea). Near-infrared fluorescence (NIRF) intensity measurements were used to evaluate the tumor accumulation of FMSN-CXCR4BPL. After 48 h post-injection, the mice were euthanized, and both tumors and major organs (liver, lungs, spleen, heart, and kidneys) were harvested. NIRF images of the excised tissues were then analyzed to determine the distribution and tumor accumulation of the treatment. All animal experiments were conducted in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines of Chonnam National University, South Korea (Approval No. CNU IACUC-H-2024-23).\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn-vivo antitumor activity\u003c/em\u003e\u003c/p\u003e\u003cp\u003eBALB/c mice were s.c. injected with 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CT26 cells into the right flank to establish tumors. Once the tumors reached an approximate volume of 100 mm\u0026sup3;, the mice were randomly assigned to one of six groups (four mice per group): PBS (control), free BBR, free PTX, a combination of BBR/PTX, FSMN(BBR/PTX), and FMSN(BBR)-CXCR4BPL(PTX). Treatments were administered intravenously, with each mouse receiving 100 \u0026micro;L of either PBS or the prepared nanoparticle formulations, corresponding to a BBR dose of 5 mg/kg, on day 0. Tumor size was measured every 2 d using Vernier calipers, and tumor volume was calculated using the equation:\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:length\\times\\:{width}^{2}\\times\\:\\frac{1}{2}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eBody weight was recorded at each measurement point. On day 21 post-treatment, the mice were euthanized, and tumors, along with major organs (heart, liver, spleen, lungs, and kidneys), were harvested. Tumors and major organs were fixed in 10% neutral buffered formalin, then paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H\u0026amp;E). The H\u0026amp;E-stained sections were subsequently examined under an inverted light microscope.\u003c/p\u003e\u003cp\u003e\u003cem\u003eImmunohistochemical analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFollowing deparaffinization, tissue slides underwent antigen retrieval through heat-mediated incubation in citrate buffer (pH 6.0) at 60\u0026deg;C for 15 min. After retrieval, sections were blocked with 5% bovine serum albumin (BSA) to prevent non-specific binding. The slides were then incubated overnight at 4\u0026deg;C with primary antibodies specific for Ki67 (catalog #ab15580) and CD31 (catalog #cell signalling 77699S), diluted in a blocking buffer containing 1% BSA in PBST (phosphate-buffered saline with Tween 20). Following primary antibody incubation, the sections were washed three times with PBST and subsequently incubated for 1 h at room temperature with secondary antibodies in PBST. Fluorescence images were acquired using a CLSM.\u003c/p\u003e\u003cp\u003e\u003cem\u003eTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTumor tissue sections were prepared for TUNEL assay to assess apoptotic cell death. The sections were first deparaffinized to remove embedding materials. Following deparaffinization, staining was conducted using the Dead End\u0026trade; Fluorometric TUNEL System (Promega) following the manufacturer\u0026rsquo;s instructions. After staining, fluorescence images were acquired and analyzed with a CLSM to evaluate the extent of apoptosis in the tumor tissues.\u003c/p\u003e\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eExperimental data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses were conducted using unpaired Student\u0026rsquo;s t-test for comparisons between two groups, one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for comparisons involving more than three groups. For comparisons of two independent variables, two-way ANOVA was used, followed by Tukey's post-hoc test. Statistical significance was determined with the following criteria: *\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/em\u003e. All analyses were performed using GraphPad Prism 6 (GraphPad Software).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Research Foundation of Korea (NRF) funded by Ministry of Science and ICT (No. RS-2024-00449435 and No. 2020R1A2C2005620).\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\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.J. conceived and designed the project, performed all AI-driven tasks, synthesized all materials, performed physicochemical characterisation, executed and analyzed all \u003cem\u003ein-vitro\u003c/em\u003e assays, curated the data, prepared the figures and tables, and wrote the manuscript. A.B. led the \u003cem\u003ein-vivo\u003c/em\u003e studies, with technical assistance from S.C and A.V. J.J.M. provided feedback throughout the study. I.K.P. and H.P. supervised the research, secured funding, and contributed to project administration. All authors approved the final version for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with the Institutional Animal Care and Use Committee Chonnam National University (CNU IACUC-H-2024-23), South Korea.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZimmermann GR, Lehar J, Keith CTJDdt: \u003cstrong\u003eMulti-target therapeutics: when the whole is greater than the sum of the parts.\u003c/strong\u003e \u003cem\u003eDrug discovery today \u003c/em\u003e2007, \u003cstrong\u003e12:\u003c/strong\u003e34-42.\u003c/li\u003e\n\u003cli\u003ePushpalatha R, Selvamuthukumar S, Kilimozhi DJJoDDS, Technology: \u003cstrong\u003eNanocarrier mediated combination drug delivery for chemotherapy\u0026ndash;A review.\u003c/strong\u003e \u003cem\u003eJournal of Drug Delivery Science and Technology \u003c/em\u003e2017, \u003cstrong\u003e39:\u003c/strong\u003e362-371.\u003c/li\u003e\n\u003cli\u003eKolishetti N, Dhar S, Valencia PM, Lin LQ, Karnik R, Lippard SJ, Langer R, Farokhzad OCJPotNAoS: \u003cstrong\u003eEngineering of self-assembled nanoparticle platform for precisely controlled combination drug therapy.\u003c/strong\u003e \u003cem\u003eProceedings of the National Academy of Sciences \u003c/em\u003e2010, \u003cstrong\u003e107:\u003c/strong\u003e17939-17944.\u003c/li\u003e\n\u003cli\u003eHu C-MJ, Zhang LJBp: \u003cstrong\u003eNanoparticle-based combination therapy toward overcoming drug resistance in cancer.\u003c/strong\u003e \u003cem\u003eBiochemical pharmacology \u003c/em\u003e2012, \u003cstrong\u003e83:\u003c/strong\u003e1104-1111.\u003c/li\u003e\n\u003cli\u003eGurunathan S, Kang M-H, Qasim M, Kim J-HJIjoms: \u003cstrong\u003eNanoparticle-mediated combination therapy: Two-in-one approach for cancer.\u003c/strong\u003e \u003cem\u003eInternational journal of molecular sciences \u003c/em\u003e2018, \u003cstrong\u003e19:\u003c/strong\u003e3264.\u003c/li\u003e\n\u003cli\u003eDevita Jr VT, Young RC, Canellos GPJC: \u003cstrong\u003eCombination versus single agent chemotherapy: a review of the basis for selection of drug treatment of cancer.\u003c/strong\u003e \u003cem\u003eCancer \u003c/em\u003e1975, \u003cstrong\u003e35:\u003c/strong\u003e98-110.\u003c/li\u003e\n\u003cli\u003eJia J, Zhu F, Ma X, Cao ZW, Li YX, Chen YZJNrDd: \u003cstrong\u003eMechanisms of drug combinations: interaction and network perspectives.\u003c/strong\u003e \u003cem\u003eNature reviews Drug discovery \u003c/em\u003e2009, \u003cstrong\u003e8:\u003c/strong\u003e111-128.\u003c/li\u003e\n\u003cli\u003eXu J, Jin B, Ren Z, Lou Y, Zhou Z, Yang Q, Han BJPo: \u003cstrong\u003eChemotherapy plus erlotinib versus chemotherapy alone for treating advanced non-small cell lung cancer: a meta-analysis.\u003c/strong\u003e \u003cem\u003ePloS one \u003c/em\u003e2015, \u003cstrong\u003e10:\u003c/strong\u003ee0131278.\u003c/li\u003e\n\u003cli\u003eXiao W, Zeng X, Lin H, Han K, Jia H-Z, Zhang X-ZJCc: \u003cstrong\u003eDual stimuli-responsive multi-drug delivery system for the individually controlled release of anti-cancer drugs.\u003c/strong\u003e \u003cem\u003eChemical communications \u003c/em\u003e2015, \u003cstrong\u003e51:\u003c/strong\u003e1475-1478.\u003c/li\u003e\n\u003cli\u003eKibria G, Hatakeyama H, Harashima HJAopr: \u003cstrong\u003eCancer multidrug resistance: mechanisms involved and strategies for circumvention using a drug delivery system.\u003c/strong\u003e \u003cem\u003eArchives of pharmacal research \u003c/em\u003e2014, \u003cstrong\u003e37:\u003c/strong\u003e4-15.\u003c/li\u003e\n\u003cli\u003eJang Y, Kim A, Moon JJ, Lee JY, Park HJAMT: \u003cstrong\u003eNovel bioengineering strategies for drug delivery systems.\u003c/strong\u003e \u003cem\u003eApplied Materials Today \u003c/em\u003e2023, \u003cstrong\u003e33:\u003c/strong\u003e101834.\u003c/li\u003e\n\u003cli\u003eMaeda H, Wu J, Sawa T, Matsumura Y, Hori KJJocr: \u003cstrong\u003eTumor vascular permeability and the EPR effect in macromolecular therapeutics: a review.\u003c/strong\u003e \u003cem\u003eJournal of controlled release \u003c/em\u003e2000, \u003cstrong\u003e65:\u003c/strong\u003e271-284.\u003c/li\u003e\n\u003cli\u003eDuncan RJNrc: \u003cstrong\u003ePolymer conjugates as anticancer nanomedicines.\u003c/strong\u003e \u003cem\u003eNature reviews cancer \u003c/em\u003e2006, \u003cstrong\u003e6:\u003c/strong\u003e688-701.\u003c/li\u003e\n\u003cli\u003eKumari P, Ghosh B, Biswas SJJodt: \u003cstrong\u003eNanocarriers for cancer-targeted drug delivery.\u003c/strong\u003e \u003cem\u003eJournal of drug targeting \u003c/em\u003e2016, \u003cstrong\u003e24:\u003c/strong\u003e179-191.\u003c/li\u003e\n\u003cli\u003eHossen S, Hossain MK, Basher M, Mia M, Rahman M, Uddin MJJJoar: \u003cstrong\u003eSmart nanocarrier-based drug delivery systems for cancer therapy and toxicity studies: A review.\u003c/strong\u003e \u003cem\u003eJournal of advanced research \u003c/em\u003e2019, \u003cstrong\u003e15:\u003c/strong\u003e1-18.\u003c/li\u003e\n\u003cli\u003eWilczewska AZ, Niemirowicz K, Markiewicz KH, Car HJPr: \u003cstrong\u003eNanoparticles as drug delivery systems.\u003c/strong\u003e \u003cem\u003ePharmacological reports \u003c/em\u003e2012, \u003cstrong\u003e64:\u003c/strong\u003e1020-1037.\u003c/li\u003e\n\u003cli\u003eXu W, Riikonen J, Lehto V-PJIjop: \u003cstrong\u003eMesoporous systems for poorly soluble drugs.\u003c/strong\u003e \u003cem\u003eInternational journal of pharmaceutics \u003c/em\u003e2013, \u003cstrong\u003e453:\u003c/strong\u003e181-197.\u003c/li\u003e\n\u003cli\u003eLin Y-S, Abadeer N, Haynes CLJCC: \u003cstrong\u003eStability of small mesoporous silica nanoparticles in biological media.\u003c/strong\u003e \u003cem\u003eChemical Communications \u003c/em\u003e2011, \u003cstrong\u003e47:\u003c/strong\u003e532-534.\u003c/li\u003e\n\u003cli\u003eTaylor KM, Kim JS, Rieter WJ, An H, Lin W, Lin WJJotACS: \u003cstrong\u003eMesoporous silica nanospheres as highly efficient MRI contrast agents.\u003c/strong\u003e \u003cem\u003eJournal of the American Chemical Society \u003c/em\u003e2008, \u003cstrong\u003e130:\u003c/strong\u003e2154-2155.\u003c/li\u003e\n\u003cli\u003eLee CH, Cheng SH, Wang YJ, Chen YC, Chen NT, Souris J, Chen CT, Mou CY, Yang CS, Lo LWJAFM: \u003cstrong\u003eNear\u003c/strong\u003e\u003cstrong\u003e‐infrared mesoporous silica nanoparticles for optical imaging: characterization and in vivo biodistribution.\u003c/strong\u003e \u003cem\u003eAdvanced Functional Materials \u003c/em\u003e2009, \u003cstrong\u003e19:\u003c/strong\u003e215-222.\u003c/li\u003e\n\u003cli\u003eLu F, Wu SH, Hung Y, Mou CYJS: \u003cstrong\u003eSize effect on cell uptake in well\u003c/strong\u003e\u003cstrong\u003e‐suspended, uniform mesoporous silica nanoparticles.\u003c/strong\u003e \u003cem\u003eSmall \u003c/em\u003e2009, \u003cstrong\u003e5:\u003c/strong\u003e1408-1413.\u003c/li\u003e\n\u003cli\u003eHe Q, Zhang Z, Gao Y, Shi J, Li YJS: \u003cstrong\u003eIntracellular localization and cytotoxicity of spherical mesoporous silica nano\u003c/strong\u003e\u003cstrong\u003e‐and microparticles.\u003c/strong\u003e \u003cem\u003eSmall \u003c/em\u003e2009, \u003cstrong\u003e5:\u003c/strong\u003e2722-2729.\u003c/li\u003e\n\u003cli\u003eHe Q, Shi JJJoMC: \u003cstrong\u003eMesoporous silica nanoparticle based nano drug delivery systems: synthesis, controlled drug release and delivery, pharmacokinetics and biocompatibility.\u003c/strong\u003e \u003cem\u003eJournal of Materials Chemistry \u003c/em\u003e2011, \u003cstrong\u003e21:\u003c/strong\u003e5845-5855.\u003c/li\u003e\n\u003cli\u003eMeka AK, Jenkins LJ, D\u0026agrave;valos-Salas M, Pujara N, Wong KY, Kumeria T, Mariadason JM, Popat AJP: \u003cstrong\u003eEnhanced solubility, permeability and anticancer activity of vorinostat using tailored mesoporous silica nanoparticles.\u003c/strong\u003e \u003cem\u003ePharmaceutics \u003c/em\u003e2018, \u003cstrong\u003e10:\u003c/strong\u003e283.\u003c/li\u003e\n\u003cli\u003eBagwe RP, Hilliard LR, Tan WJL: \u003cstrong\u003eSurface modification of silica nanoparticles to reduce aggregation and nonspecific binding.\u003c/strong\u003e \u003cem\u003eLangmuir \u003c/em\u003e2006, \u003cstrong\u003e22:\u003c/strong\u003e4357-4362.\u003c/li\u003e\n\u003cli\u003eSantra S, Liesenfeld B, Bertolino C, Dutta D, Cao Z, Tan W, Moudgil BM, Mericle RAJJoL: \u003cstrong\u003eFluorescence lifetime measurements to determine the core\u0026ndash;shell nanostructure of FITC-doped silica nanoparticles: An optical approach to evaluate nanoparticle photostability.\u003c/strong\u003e \u003cem\u003eJournal of Luminescence \u003c/em\u003e2006, \u003cstrong\u003e117:\u003c/strong\u003e75-82.\u003c/li\u003e\n\u003cli\u003eZhang Q, Wang J, Zhang H, Liu D, Ming L, Liu L, Dong Y, Jian B, Cai DJRa: \u003cstrong\u003eThe anticancer efficacy of paclitaxel liposomes modified with low-toxicity hydrophobic cell-penetrating peptides in breast cancer: an in vitro and in vivo evaluation.\u003c/strong\u003e \u003cem\u003eRSC advances \u003c/em\u003e2018, \u003cstrong\u003e8:\u003c/strong\u003e24084-24093.\u003c/li\u003e\n\u003cli\u003eDeshpande PP, Biswas S, Torchilin VPJN: \u003cstrong\u003eCurrent trends in the use of liposomes for tumor targeting.\u003c/strong\u003e \u003cem\u003eNanomedicine \u003c/em\u003e2013, \u003cstrong\u003e8:\u003c/strong\u003e1509-1528.\u003c/li\u003e\n\u003cli\u003eDerycke AS, Kamuhabwa A, Gijsens A, Roskams T, De Vos D, Kasran A, Huwyler J, Missiaen L, de Witte PAJJotNCI: \u003cstrong\u003eTransferrin-conjugated liposome targeting of photosensitizer AlPcS 4 to rat bladder carcinoma cells.\u003c/strong\u003e \u003cem\u003eJournal of the National Cancer Institute \u003c/em\u003e2004, \u003cstrong\u003e96:\u003c/strong\u003e1620-1630.\u003c/li\u003e\n\u003cli\u003eNoble GT, Stefanick JF, Ashley JD, Kiziltepe T, Bilgicer BJTib: \u003cstrong\u003eLigand-targeted liposome design: challenges and fundamental considerations.\u003c/strong\u003e \u003cem\u003eTrends in biotechnology \u003c/em\u003e2014, \u003cstrong\u003e32:\u003c/strong\u003e32-45.\u003c/li\u003e\n\u003cli\u003eXu J, Long Y, Ni L, Yuan X, Yu N, Wu R, Tao J, Zhang YJBc: \u003cstrong\u003eAnticancer effect of berberine based on experimental animal models of various cancers: a systematic review and meta-analysis.\u003c/strong\u003e \u003cem\u003eBMC cancer \u003c/em\u003e2019, \u003cstrong\u003e19:\u003c/strong\u003e1-20.\u003c/li\u003e\n\u003cli\u003eLiu H, Huang C, Wu L, Wen BJO, therapy: \u003cstrong\u003eEffect of evodiamine and berberine on miR-429 as an oncogene in human colorectal cancer.\u003c/strong\u003e \u003cem\u003eOncoTargets and therapy \u003c/em\u003e2016\u003cstrong\u003e:\u003c/strong\u003e4121-4127.\u003c/li\u003e\n\u003cli\u003eKim S, Lee SY, Cho H-JJB, communications br: \u003cstrong\u003eBerberine and zinc oxide-based nanoparticles for the chemo-photothermal therapy of lung adenocarcinoma.\u003c/strong\u003e \u003cem\u003eBiochemical and biophysical research communications \u003c/em\u003e2018, \u003cstrong\u003e501:\u003c/strong\u003e765-770.\u003c/li\u003e\n\u003cli\u003eTian Y, Zhao L, Wang Y, Zhang H, Xu D, Zhao X, Li Y, Li JJAjoa: \u003cstrong\u003eBerberine inhibits androgen synthesis by interaction with aldo-keto reductase 1C3 in 22Rv1 prostate cancer cells.\u003c/strong\u003e \u003cem\u003eAsian journal of andrology \u003c/em\u003e2016, \u003cstrong\u003e18:\u003c/strong\u003e607.\u003c/li\u003e\n\u003cli\u003eWu K, Yang Q, Mu Y, Zhou L, Liu Y, Zhou Q, He BJIjoo: \u003cstrong\u003eBerberine inhibits the proliferation of colon cancer cells by inactivating Wnt/\u0026beta;-catenin signaling.\u003c/strong\u003e \u003cem\u003eInternational journal of oncology \u003c/em\u003e2012, \u003cstrong\u003e41:\u003c/strong\u003e292-298.\u003c/li\u003e\n\u003cli\u003eSu Y-H, Tang W-C, Cheng Y-W, Sia P, Huang C-C, Lee Y-C, Jiang H-Y, Wu M-H, Lai I-L, Lee J-WJBeBA-MCR: \u003cstrong\u003eTargeting of multiple oncogenic signaling pathways by Hsp90 inhibitor alone or in combination with berberine for treatment of colorectal cancer.\u003c/strong\u003e \u003cem\u003eBiochimica et Biophysica Acta (BBA)-Molecular Cell Research \u003c/em\u003e2015, \u003cstrong\u003e1853:\u003c/strong\u003e2261-2272.\u003c/li\u003e\n\u003cli\u003eAlmatroodi SA, Alsahli MA, Rahmani AHJM: \u003cstrong\u003eBerberine: An important emphasis on its anticancer effects through modulation of various cell signaling pathways.\u003c/strong\u003e \u003cem\u003eMolecules \u003c/em\u003e2022, \u003cstrong\u003e27:\u003c/strong\u003e5889.\u003c/li\u003e\n\u003cli\u003eJiang X, Jiang Z, Jiang M, Sun Y: \u003cstrong\u003eBerberine as a potential agent for the treatment of colorectal cancer.\u003c/strong\u003e \u003cem\u003eFrontiers in Medicine \u003c/em\u003e2022, \u003cstrong\u003e9:\u003c/strong\u003e886996.\u003c/li\u003e\n\u003cli\u003eGe X, Lee Y-T, Yeh S-J: \u003cstrong\u003eMD-Syn: Synergistic drug combination prediction based on the multidimensional feature fusion method and attention mechanisms.\u003c/strong\u003e \u003cem\u003earXiv preprint arXiv:250107884 \u003c/em\u003e2025.\u003c/li\u003e\n\u003cli\u003eSmitha K, Anitha A, Furuike T, Tamura H, Nair SV, Jayakumar RJC, Biointerfaces SB: \u003cstrong\u003eIn vitro evaluation of paclitaxel loaded amorphous chitin nanoparticles for colon cancer drug delivery.\u003c/strong\u003e \u003cem\u003eColloids and Surfaces B: Biointerfaces \u003c/em\u003e2013, \u003cstrong\u003e104:\u003c/strong\u003e245-253.\u003c/li\u003e\n\u003cli\u003eAbu Samaan TM, Samec M, Liskova A, Kubatka P, B\u0026uuml;sselberg DJB: \u003cstrong\u003ePaclitaxel\u0026rsquo;s mechanistic and clinical effects on breast cancer.\u003c/strong\u003e \u003cem\u003eBiomolecules \u003c/em\u003e2019, \u003cstrong\u003e9:\u003c/strong\u003e789.\u003c/li\u003e\n\u003cli\u003eArmstrong DK, Bundy B, Wenzel L, Huang HQ, Baergen R, Lele S, Copeland LJ, Walker JL, Burger RAJNEJoM: \u003cstrong\u003eIntraperitoneal cisplatin and paclitaxel in ovarian cancer.\u003c/strong\u003e \u003cem\u003eNew England Journal of Medicine \u003c/em\u003e2006, \u003cstrong\u003e354:\u003c/strong\u003e34-43.\u003c/li\u003e\n\u003cli\u003eJim\u0026eacute;nez-L\u0026oacute;pez J, El-Hammadi MM, Ortiz R, Cayero-Otero MD, Cabeza L, Perazzoli G, Martin-Banderas L, Baeyens JM, Prados J, Melguizo CJPR: \u003cstrong\u003eA novel nanoformulation of PLGA with high non-ionic surfactant content improves in vitro and in vivo PTX activity against lung cancer.\u003c/strong\u003e \u003cem\u003ePharmacological Research \u003c/em\u003e2019, \u003cstrong\u003e141:\u003c/strong\u003e451-465.\u003c/li\u003e\n\u003cli\u003eSun J, Jiang L, Lin Y, Gerhard EM, Jiang X, Li L, Yang J, Gu ZJIjon: \u003cstrong\u003eEnhanced anticancer efficacy of paclitaxel through multistage tumor-targeting liposomes modified with RGD and KLA peptides.\u003c/strong\u003e \u003cem\u003eInternational journal of nanomedicine \u003c/em\u003e2017\u003cstrong\u003e:\u003c/strong\u003e1517-1537.\u003c/li\u003e\n\u003cli\u003eMa P, Chen J, Bi X, Li Z, Gao X, Li H, Zhu H, Huang Y, Qi J, Zhang YJAam, interfaces: \u003cstrong\u003eOvercoming multidrug resistance through the GLUT1-mediated and enzyme-triggered mitochondrial targeting conjugate with redox-sensitive paclitaxel release.\u003c/strong\u003e \u003cem\u003eACS applied materials \u0026amp; interfaces \u003c/em\u003e2018, \u003cstrong\u003e10:\u003c/strong\u003e12351-12363.\u003c/li\u003e\n\u003cli\u003eMurray S, Briasoulis E, Linardou H, Bafaloukos D, Papadimitriou CJCtr: \u003cstrong\u003eTaxane resistance in breast cancer: mechanisms, predictive biomarkers and circumvention strategies.\u003c/strong\u003e \u003cem\u003eCancer treatment reviews \u003c/em\u003e2012, \u003cstrong\u003e38:\u003c/strong\u003e890-903.\u003c/li\u003e\n\u003cli\u003eCheng Y, Ji YJJocr: \u003cstrong\u003eMitochondria-targeting nanomedicine self-assembled from GSH-responsive paclitaxel-ss-berberine conjugate for synergetic cancer treatment with enhanced cytotoxicity.\u003c/strong\u003e \u003cem\u003eJournal of controlled release \u003c/em\u003e2020, \u003cstrong\u003e318:\u003c/strong\u003e38-49.\u003c/li\u003e\n\u003cli\u003eXu C, Zheng L, Li D, Chen G, Gu J, Chen J, Yao Q: \u003cstrong\u003eCXCR4 overexpression is correlated with poor prognosis in colorectal cancer.\u003c/strong\u003e \u003cem\u003eLife sciences \u003c/em\u003e2018, \u003cstrong\u003e208:\u003c/strong\u003e333-340.\u003c/li\u003e\n\u003cli\u003eKollmar O, Rupertus K, Scheuer C, Nickels RM, Haberl GC, Tilton B, Menger MD, Schilling MK: \u003cstrong\u003eCXCR4 and CXCR7 regulate angiogenesis and CT26. WT tumor growth independent from SDF\u003c/strong\u003e\u003cstrong\u003e‐1.\u003c/strong\u003e \u003cem\u003eInternational journal of cancer \u003c/em\u003e2010, \u003cstrong\u003e126:\u003c/strong\u003e1302-1315.\u003c/li\u003e\n\u003cli\u003eKurcinski M, Jamroz M, Blaszczyk M, Kolinski A, Kmiecik S: \u003cstrong\u003eCABS-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site.\u003c/strong\u003e \u003cem\u003eNucleic acids research \u003c/em\u003e2015, \u003cstrong\u003e43:\u003c/strong\u003eW419-W424.\u003c/li\u003e\n\u003cli\u003eInc N: \u003cstrong\u003eNeurosnap: An online platform for computational biology and chemistry.\u003c/strong\u003e 2022.\u003c/li\u003e\n\u003cli\u003eBrand S, Dambacher J, Beigel F, Olszak T, Diebold J, Otte J-M, G\u0026ouml;ke B, Eichhorst STJEcr: \u003cstrong\u003eCXCR4 and CXCL12 are inversely expressed in colorectal cancer cells and modulate cancer cell migration, invasion and MMP-9 activation.\u003c/strong\u003e \u003cem\u003eExperimental cell research \u003c/em\u003e2005, \u003cstrong\u003e310:\u003c/strong\u003e117-130.\u003c/li\u003e\n\u003cli\u003eShin H-N, Moon H-H, Ku J-LJIjomm: \u003cstrong\u003eStromal cell-derived factor-1\u0026alpha; and macrophage migration-inhibitory factor induce metastatic behavior in CXCR4-expressing colon cancer cells.\u003c/strong\u003e \u003cem\u003eInternational journal of molecular medicine \u003c/em\u003e2012, \u003cstrong\u003e30:\u003c/strong\u003e1537-1543.\u003c/li\u003e\n\u003cli\u003eEgorova A, Kiselev A, Hakli M, Ruponen M, Baranov V, Urtti AJTJoGMAcdjfrotsogt, applications ic: \u003cstrong\u003eChemokine\u003c/strong\u003e\u003cstrong\u003e‐derived peptides as carriers for gene delivery to CXCR4 expressing cells.\u003c/strong\u003e \u003cem\u003eThe Journal of Gene Medicine: A cross\u003c/em\u003e\u003cem\u003e‐disciplinary journal for research on the science of gene transfer and its clinical applications \u003c/em\u003e2009, \u003cstrong\u003e11:\u003c/strong\u003e772-781.\u003c/li\u003e\n\u003cli\u003eDubey R, Rajesh Y, More MJMTP: \u003cstrong\u003eSynthesis and characterization of SiO2 nanoparticles via sol-gel method for industrial applications.\u003c/strong\u003e \u003cem\u003eMaterials Today: Proceedings \u003c/em\u003e2015, \u003cstrong\u003e2:\u003c/strong\u003e3575-3579.\u003c/li\u003e\n\u003cli\u003eJiang X, Tang X, Tang L, Zhang B, Mao HJCI: \u003cstrong\u003eSynthesis and formation mechanism of amorphous silica particles via sol\u0026ndash;gel process with tetraethylorthosilicate.\u003c/strong\u003e \u003cem\u003eCeramics International \u003c/em\u003e2019, \u003cstrong\u003e45:\u003c/strong\u003e7673-7680.\u003c/li\u003e\n\u003cli\u003eChaudhary Z, Khan GM, Abeer MM, Pujara N, Tse BW-C, McGuckin MA, Popat A, Kumeria TJBs: \u003cstrong\u003eEfficient photoacoustic imaging using indocyanine green (ICG) loaded functionalized mesoporous silica nanoparticles.\u003c/strong\u003e \u003cem\u003eBiomaterials science \u003c/em\u003e2019, \u003cstrong\u003e7:\u003c/strong\u003e5002-5015.\u003c/li\u003e\n\u003cli\u003eLiu J, Jiang X, Ashley C, Brinker CJJJotACS: \u003cstrong\u003eElectrostatically mediated liposome fusion and lipid exchange with a nanoparticle-supported bilayer for control of surface charge, drug containment, and delivery.\u003c/strong\u003e \u003cem\u003eJournal of the American Chemical Society \u003c/em\u003e2009, \u003cstrong\u003e131:\u003c/strong\u003e7567-7569.\u003c/li\u003e\n\u003cli\u003eDong J, Wu Z, Xu H, Ouyang D: \u003cstrong\u003eFormulationAI: a novel web-based platform for drug formulation design driven by artificial intelligence.\u003c/strong\u003e \u003cem\u003eBriefings in Bioinformatics \u003c/em\u003e2023, \u003cstrong\u003e25\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN: \u003cstrong\u003eInvolvement of chemokine receptors in breast cancer metastasis.\u003c/strong\u003e \u003cem\u003enature \u003c/em\u003e2001, \u003cstrong\u003e410:\u003c/strong\u003e50-56.\u003c/li\u003e\n\u003cli\u003eWang Y, Wang Q, Wang X, Yao P, Dai Q, Qi X, Yang M, Zhang X, Huang R, Yang J, et al: \u003cstrong\u003eDocetaxel-loaded pH/ROS dual-responsive nanoparticles with self-supplied ROS for inhibiting metastasis and enhancing immunotherapy of breast cancer.\u003c/strong\u003e \u003cem\u003eJournal of Nanobiotechnology \u003c/em\u003e2023, \u003cstrong\u003e21:\u003c/strong\u003e286.\u003c/li\u003e\n\u003cli\u003eLu J, Liong M, Zink JI, Tamanoi FJs: \u003cstrong\u003eMesoporous silica nanoparticles as a delivery system for hydrophobic anticancer drugs.\u003c/strong\u003e \u003cem\u003esmall \u003c/em\u003e2007, \u003cstrong\u003e3:\u003c/strong\u003e1341-1346.\u003c/li\u003e\n\u003cli\u003eSlowing I, Trewyn BG, Lin VS-YJJotACS: \u003cstrong\u003eEffect of surface functionalization of MCM-41-type mesoporous silica nanoparticles on the endocytosis by human cancer cells.\u003c/strong\u003e \u003cem\u003eJournal of the American Chemical Society \u003c/em\u003e2006, \u003cstrong\u003e128:\u003c/strong\u003e14792-14793.\u003c/li\u003e\n\u003cli\u003eHuh S, Wiench JW, Yoo J-C, Pruski M, Lin VS-YJCom: \u003cstrong\u003eOrganic functionalization and morphology control of mesoporous silicas via a co-condensation synthesis method.\u003c/strong\u003e \u003cem\u003eChemistry of materials \u003c/em\u003e2003, \u003cstrong\u003e15:\u003c/strong\u003e4247-4256.\u003c/li\u003e\n\u003cli\u003eHansen CB, Kao GY, Moase EH, Zalipsky S, Allen TMJBeBA-B: \u003cstrong\u003eAttachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures.\u003c/strong\u003e 1995, \u003cstrong\u003e1239:\u003c/strong\u003e133-144.\u003c/li\u003e\n\u003cli\u003eXu Y, Meng HJAC, Nanomedicine,, Biotechnology: \u003cstrong\u003ePaclitaxel-loaded stealth liposomes: development, characterization, pharmacokinetics, and biodistribution.\u003c/strong\u003e \u003cem\u003eArtificial Cells, Nanomedicine, and Biotechnology \u003c/em\u003e2016, \u003cstrong\u003e44:\u003c/strong\u003e350-355.\u003c/li\u003e\n\u003cli\u003eRuttala HB, Ko YTJC, Biointerfaces SB: \u003cstrong\u003eLiposomal co-delivery of curcumin and albumin/paclitaxel nanoparticle for enhanced synergistic antitumor efficacy.\u003c/strong\u003e\u003cem\u003eColloids and Surfaces B: Biointerfaces \u003c/em\u003e2015, \u003cstrong\u003e128:\u003c/strong\u003e419-426.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"AI-guided nanomedicine, CXCR4-targeted core–shell, mesoporous silica nanoparticle, liposome coating, combination chemotherapy","lastPublishedDoi":"10.21203/rs.3.rs-7307122/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7307122/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAn integrated three-step artificial-intelligence (AI) workflow was used to accelerate the design of a CXCR4-targeted, dual-drug nanocarrier for colorectal-cancer therapy. First, the MD-Syn platform screened 38 approved oncology agents across six human colorectal-cancer cell lines (HCT116, HT29, LoVo, RKO, SW620, SW837) and identified paclitaxel (PTX) as the compound with a\u0026thinsp;\u0026gt;\u0026thinsp;0.9999 probability of synergy with (BBR). Second, a dual peptide-discovery pipeline that combined CABS-dock flexible docking with AlphaFold-multimer/NeuroSNAP-AI scoring yielded a high-affinity 9-mer stromal-cell-derived factor-1 fragment (CXCR4-binding peptide, CXCR4BP; ipTM\u0026thinsp;=\u0026thinsp;0.95). The peptide was conjugated to DSPE-PEG₂₀₀₀ to form a targeting liposome (CXCR4BPL). Third, FormulationAI predicted that loading PTX into the CXCR4BPL shell and BBR into phosphonate-functionalized mesoporous silica nanoparticles (FMSNs) would maximize encapsulation. A one-pot self-assembly gave FMSN(BBR)-CXCR4BPL(PTX) core\u0026ndash;shell nanocarrier (~\u0026thinsp;130 nm) with experimental entrapment efficiencies of 78.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9% for BBR and 75.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4% for PTX and sustained co-release over 72 h at pH 7.4. In CXCR4-positive CT26 cells, the core-shell nanocarrier curtailed scratch-wound closure by ~\u0026thinsp;94% and reduced metabolic viability to 36% at 72 h, markedly outperforming free drugs and non-targeted controls. A single intravenous dose delivering 5 mg/kg BBR equivalent in CT26-bearing mice restricted tumor volume and mass to ~\u0026thinsp;15% and ~\u0026thinsp;13% of PBS controls, normalized splenomegaly four-fold, and produced pronounced decreases in Ki-67 and CD31 with increased TUNEL positivity, all without body-weight loss or organ pathology. These results demonstrate that AI-guided synergy scouting, ligand discovery and drug-allocation modelling can be seamlessly combined with modular nanocarrier engineering to generate a high-payload, CXCR4-targeted PTX/BBR therapy that delivers potent antitumor efficacy alongside excellent systemic tolerance, offering a transferable blueprint for next-generation combination nanomedicines.\u003c/p\u003e","manuscriptTitle":"AI-guided design of a CXCR4-targeted core–shell nanocarrier for co-delivery of berberine/paclitaxel in cancer therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-14 11:17:25","doi":"10.21203/rs.3.rs-7307122/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-08T08:44:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-05T03:55:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-01T02:26:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75080451065710400446362036837312617941","date":"2025-08-12T02:07:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185085740249381457256275225526815064931","date":"2025-08-11T01:15:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-09T08:59:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-06T08:40:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-06T08:40:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-08-06T07:51:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bd016c56-3f44-4775-8fc7-0f413d748205","owner":[],"postedDate":"August 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-25T23:31:19+00:00","versionOfRecord":{"articleIdentity":"rs-7307122","link":"https://doi.org/10.1186/s12951-025-03850-9","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2025-11-22 15:58:31","publishedOnDateReadable":"November 22nd, 2025"},"versionCreatedAt":"2025-08-14 11:17:25","video":"","vorDoi":"10.1186/s12951-025-03850-9","vorDoiUrl":"https://doi.org/10.1186/s12951-025-03850-9","workflowStages":[]},"version":"v1","identity":"rs-7307122","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7307122","identity":"rs-7307122","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}