Design and Characterization of Peptide-Conjugated Solid Lipid Nanoparticles for Targeted MRI and SPECT Imaging of Breast Tumors

Abstract

Triple-negative breast cancer (TNBC) presents significant challenges due to its aggressive behavior and lack of targeted treatments. High-resolution imaging techniques and targeted nanoparticles offer potential solutions for early detection and monitoring of TNBC. In this study, we developed and characterized solid lipid nanoparticles (SLNs) conjugated with a C-peptide derived from endostatin to target integrin αvβ₃, overexpressed in TNBC. These SLNs were loaded with superparamagnetic iron oxide nanoparticles (SPIONs) for enhanced magnetic resonance imaging (MRI) and radiolabeled with technetium-99m (^99mTc) for single-photon emission computed tomography (SPECT), enabling dual-modality imaging. Extensive characterization of the nanoparticles was performed utilizing a variety of advanced techniques, including dynamic light scattering (DLS), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray diffraction (XRD), vibrating sample magnetometry (VSM), field-emission scanning electron microscopy (FE-SEM), and atomic force microscopy (AFM). This comprehensive analysis validated the successful synthesis and functionalization of the nanoparticles, along with their remarkable magnetic properties, while also revealing their distinct spherical morphology, optimal size, uniform distribution, and colloidal stability. The conjugation of C-peptide significantly enhanced the targeting efficiency in vitro, as evidenced by the MTT and receptor-binding assays in 4T1 cells using flow cytometry and MRI. In vivo studies using a 4T1 murine model demonstrated that peptide-conjugated SLNs accumulated in tumor tissues, providing superior contrast in MRI and enhanced tumor-specific localization in SPECT imaging. Biodistribution analysis confirmed reduced off-target accumulation, particularly in the liver, compared to nontargeted formulations. Collectively, C-peptide-conjugated SLNs provide a promising dual-modality imaging platform for TNBC, offering improved diagnostic accuracy and tumor targeting.

Figure 1

Characterization of nanoparticles. (a) ζ-Potential, mean size, and PDI of nanoparticles assessed using DLS; see Figures S2 and S3 for details. (b) FTIR spectra (500–4000 cm^–1) revealing functional groups in nanoparticles. (c) DSC analysis, demonstrating the thermal properties of SLN and SLN-OA-Fe₃O₄. (d) TGA curves illustrating the thermal stability of the nanoparticles. (e) XRD analysis indicating the crystal structure and phase purity of the nanoparticles. (f) Magnetic properties of the nanoparticles assessed by vibrating sample magnetometry (VSM).

Figure 2

Morphological characterization of Nanoparticles. AFM images show spherical and homogeneous nanoparticles. Scale bar: 500 nm (n = 3).

Figure 3

Cell viability. (a) Viability of 4T1MCT cells treated with different concentrations of free peptide (0.5, 1, 2, 4, 6, 8, 10, 12, 14, and 16 μg·mL^–1) after 24 h. (b) Viability of 4T1MCT cells treated with SLN, OA-Fe₃O₄, SLN-OA-Fe₃O₄, and peptide-SLN-OA-Fe₃O₄ at different concentrations over 24 h. (c) Viability of MCF10 normal cells treated with different concentrations of free peptide (0.5, 1, 2, 4, 6, 8, 10, 12, 14, and 16 μg·mL^–1) after 24 h. (d) Viability of MCF10 normal cells treated with SLN, OA-Fe₃O₄, SLN-OA-Fe₃O₄, and peptide-SLN-OA-Fe₃O₄ at different concentrations over 24 h. Cell viability was measured through the MTT assay, and data were analyzed with Prism 8 software (two-way ANOVA, mean ± SEM, n = 3, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, or ns: not significant).

Figure 4

Binding specificity studies. (a) Receptor-binding assays using flow cytometry. 4T1MCT cells were incubated with free peptide (7 μg·mL^–1) and peptide conjugated on SLN (peptide-SLN-OA-Fe₃O₄) at different concentrations (0, 2, 4, and 6 μg·mL^–1). After adding the anti-integrin αv antibody and FITC-conjugated secondary antibody, flow cytometric analysis was performed. (b) Quantitative analysis of the flow cytometry assay using Prism 8 software (two-way ANOVA, mean ± SEM, n = 3, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, or ns: not significant). (c) Evaluation of the targeting capability of peptide-SLN-OA-Fe₃O₄ using MRI. Harvested 4T1 cells were incubated with peptide-SLN-OA-Fe₃O₄ and varying doses of anti-integrin αv antibody (X = 100 ng/mL). MR images show a decreased T₂ signal in 4T1 cell phantoms by increasing doses of the anti-integrin αv antibody. (d) Quantitative analysis was performed using ImageJ and Prism 8 software (two-way ANOVA, mean ± SEM, n = 3, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, or ns: not significant).

Figure 5

Binding and targeting efficacy through MR imaging. (a) Schematic figure of in vivo MR imaging protocols. (b) T2-weighted MR images of increasing Fe₃O₄ concentration loading into SLN (μg·mL^–1) (n = 3). (c) Contrast intensity of T2-weighted MR images quantified using ImageJ software (n = 3). (d) In vivo MRI of 4T1MCT-bearing Balb-c mice. MR images were taken at various intervals following the tail vein injection of SLN-OA-Fe₃O₄ and peptide-SLN-OA-Fe₃O₄ (n = 3).

Figure 6

In vivo SPECT imaging. (a) Schematic of radiolabeling and in vivo SPECT imaging protocols. (b) 3D SPECT imaging of 4T1MCT-bearing Balb/c mice showing that ^99mTc-peptide-SLN-OA-Fe₃O₄ accumulated effectively in tumors compared to ^99mTc-SLN-OA-Fe₃O₄. Free ^99mTc exhibited a short half-life, rapidly excreting into the bladder. (c) Tissue distribution of free ^99mTc, ^99mTc-SLN-OA-Fe₃O₄, and ^99mTc-peptide-SLN-OA-Fe₃O₄ in treated mice measured by a dose calibrator after their sacrifice. The data were analyzed by two-way ANOVA using Prism software (mean ± SEM, n = 3, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, or ns: not significant).

Figure 7

Biodistribution study. (a) Prussian blue staining was performed on tumor and liver tissues from 4T1MCT-bearing BALB/c mice at 30, 60, and 180 min postinjection of SLN-OA-Fe₃O₄ and peptide-SLN-OA-Fe₃O₄; scale bar: 20 μm. Significant iron particle accumulation was observed in the tumors of peptide-SLN-OA-Fe₃O₄-treated mice, increasing over time, while SLN-OA-Fe₃O₄-treated mice showed minimal uptake. Additionally, the peptide formulation resulted in lower liver accumulation compared to SLN-OA-Fe₃O₄. (b) Microscopy analysis using ImageJ quantified the iron particles, and data were analyzed with two-way ANOVA using Prism software (mean ± SEM, n = 3, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, or ns: not significant).

Figure 8

(a) Microscopic view of Prussian blue staining, scale bar: 20 μm. (b) Quantitative analysis of iron accumulation in the lung, kidney, spleen, and heart of treated mice. Data were analyzed using two-way ANOVA using Prism software (mean ± SEM, n = 3, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, or ns: not significant).

Figure 9

H&E staining of tissues from SLN-OA-Fe₃O₄- and peptide-SLN-OA-Fe₃O₄-treated mice for biosafety evaluation (n = 3); scale bar: 20 μm. This analysis assesses the histopathological effects and biocompatibility of the nanoparticle formulations.