Dual‑Target Magnetic Nanoparticles Enable High‑Purity Lymphatic Endothelial Cell Isolation and Dual‑Modality Imaging in Colorectal Cancer

Abstract

Malignant tumors remain a leading cause of mortality worldwide. Lymphatic vessel density correlates strongly with metastasis, yet current markers lack specificity and affordability. Here we report Fe₃O₄@KCTS core‑shell magnetic nanoparticles functionalized with both LYVE‑1 and podoplanin antibodies. The resulting dual‑target probes efficiently capture lymphatic endothelial cells (LECs) from human colorectal tumors, enabling high‑purity isolation and in vivo T₂‑weighted MRI plus fluorescence imaging. These findings lay the groundwork for early clinical diagnosis and therapeutic targeting of LEC‑mediated metastasis.

Introduction

While the tumor microenvironment drives metastasis, the exact contribution of lymphatic endothelial cells (LECs) remains poorly defined. LEC‑specific markers—LYVE‑1, podoplanin, VEGFR‑3, Prox‑1—have accelerated research, but none are fully exclusive to LECs. LYVE‑1 is absent on vascular endothelium, and podoplanin is not expressed by blood vessels, making the pair highly specific. Magnetic iron oxide nanoparticles (Fe₃O₄) are biocompatible, highly paramagnetic, and amenable to surface functionalization. Chitosan derivatives, such as α‑ketoglutarate carboxymethyl chitosan (KCTS), provide abundant carboxyl groups for covalent coupling, enhancing dispersion and reducing aggregation.

Combining these attributes, we engineered Fe₃O₄@KCTS nanoparticles bearing both LYVE‑1 and podoplanin antibodies. The dual‑target design improves LEC capture fidelity, reduces contaminating vascular cells, and supports dual‑mode imaging, advancing both diagnostic and research applications.

Materials and Methods

Cell Culture

Human colorectal carcinoma HT29 cells (ATCC) were maintained at 37 °C in DMEM supplemented with 10 % FBS, 100 U mL^‑1 penicillin, and 100 µg mL^‑1 streptomycin, under 5 % CO₂.

Animal Model

Forty NOD/SCID female mice received subcutaneous HT29 injections (2 × 10⁷ cells mL^‑1, 200 µL per mouse). Protocols were approved by the Guangxi Medical University Ethics Committee.

Nanoparticle Synthesis

Fe₃O₄ cores were activated with EDC/NHS, then conjugated to KCTS via COOH–OH linkage. LYVE‑1 (APC) and podoplanin (FITC) antibodies were covalently coupled, yielding Fe₃O₄@KCTS‑LECs‑Double‑Ab (0.1 mg mL^‑1). Nanoparticles were stored at 4 °C in darkness.

Characterization

Transmission electron microscopy (TEM) revealed uniform spherical cores (≈ 90 nm). Dynamic light scattering (DLS) showed an average diameter of 91.3 ± 2.3 nm and a zeta potential of –32.8 ± 1.5 mV, indicating excellent colloidal stability. Fluorescence spectra confirmed distinct FITC (488 nm) and APC (545 nm) peaks, confirming successful antibody attachment.

Immunohistochemistry & Immunofluorescence

Frozen tumor sections were stained with LYVE‑1 (APC) and podoplanin (FITC) antibodies, counterstained with DAPI, and examined by confocal microscopy.

LEC Isolation

Tumor tissue was enzymatically dissociated, filtered, and subjected to either (1) commercial LYVE‑1 MicroBeads or (2) the Fe₃O₄@KCTS‑LECs‑Double‑Ab magnetic separation. Sorted cells were cultured in six‑well plates.

Functional Assays

Tube formation was assessed in Matrigel, and LDL uptake was quantified with DiI‑Ac‑LDL. Cell viability was measured via CCK‑8 after exposure to nanoparticle concentrations ranging from 0.1 to 2 mg mL^‑1. In vivo toxicity was evaluated by histopathology of major organs after a single 2 mg mL^‑1 injection.

Imaging Studies

NOD/SCID mice bearing subcutaneous HT29 xenografts received 0.5 mmol kg^‑1 of the dual‑target nanoparticles intravenously. T₂-weighted MRI (3.0 T, 80 × 80 mm FOV, 69 ms TE) and fluorescence imaging were performed pre‑injection and at 0.5, 12, and 24 h post‑injection. A blocking group received anti‑LYVE‑1 and anti‑podoplanin antibodies prior to nanoparticle injection.

Results

Nanoparticle Design

Scheme 1 illustrates the core‑shell construction and dual‑antibody functionalization. TEM and DLS data confirm monodispersity (91 nm) and negative surface charge (–32.8 mV). Fluorescence spectra validate co‑conjugation of FITC and APC.

LEC Purity

Flow cytometry showed 71.2 % LYVE‑1⁺/podoplanin⁻ cells with MicroBeads, versus 88.9 % double‑positive cells with the dual‑target nanoprobe (P < 0.05). Immunofluorescence confirmed co‑expression of both markers in the nanoprobe‑sorted population.

Functional Competence

Nanoprobe‑sorted LECs exhibited superior tube‑forming ability and enhanced LDL uptake relative to MicroBead‑sorted cells, indicating higher purity and functionality.

In Vivo Imaging

Fluorescence peaked at 12 h post‑injection and declined by 24 h, whereas MRI signal intensity decreased by 12 h before gradually recovering. The blocking group showed negligible tumor uptake, confirming target specificity.

Toxicity

CCK‑8 assays revealed > 90 % viability across all nanoparticle concentrations at 24 and 48 h. Histology of heart, lung, liver, spleen, and kidney showed no necrosis or inflammation after a 2 mg mL^‑1 dose.

Discussion

Dual antibody functionalization markedly improves LEC isolation, overcoming limitations of single‑marker systems that often co‑capture vascular endothelial cells. The robust negative zeta potential and uniform morphology of Fe₃O₄@KCTS‑LECs‑Double‑Ab confer stability and prevent aggregation. The dual‑modality imaging capability offers a powerful platform for early colorectal cancer detection and for studying LEC‑mediated metastasis.

Conclusions

We have engineered a biocompatible, dual‑target magnetic nanoparticle that efficiently isolates high‑purity LECs and enables simultaneous MRI and fluorescence imaging in vivo. This platform offers a cost‑effective, time‑saving alternative to conventional isolation methods and holds promise for clinical translation in colorectal cancer diagnosis and research into lymphatic metastasis mechanisms.