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. 2026 Oct 19. Online ahead of print. doi: 10.1039/d6na00729e

Peptide-functionalized gadolinium liposomes enable HER2-targeted molecular MRI of breast cancer

Serena Monti a,†, Enrico Gallo b,†, Ylenia Ferrara a, Valentina Verdoliva c, Luca Licenziato a, Cristina Terlizzi a, Annachiara Sarnella a, Sandra Albanese a, Carmen Avagliano d, Antonella Accardo e, Adelaide Greco d,f,✉, Stefania De Luca g,✉
PMCID: PMC13691995  PMID: 42921217

Abstract

Targeted molecular magnetic resonance imaging (MRI) of HER2-positive breast cancer remains challenging because of the limited sensitivity of conventional gadolinium-based contrast agents. Herein, we developed multifunctional gadolinium-loaded liposomes functionalized with A9-derived HER2-targeting peptides to achieve selective tumor recognition and enhanced MRI performance. The liposomes exhibited good colloidal stability, enhanced relaxivity compared with free Gd-DTPA, and negligible cytotoxicity. Surface functionalization significantly increased binding to HER2-overexpressing BT-474 cells, with peptide-functionalized liposomes showing the highest affinity. In a preliminary HER2-positive breast cancer xenograft model, peptide-targeted liposomes showed tumor uptake and sustained MRI contrast enhancement, whereas non-functionalized liposomes showed lower tumour contrast enhancement. These preliminary observations support further investigation of peptide-functionalized gadolinium liposomes as a promising molecular MRI platform for HER2-targeted imaging and future theranostic applications.

A HER2-targeted selective liposomal platform, incorporating an amphiphilic Gd(iii) MRI-CA and externally derivatized with A9 peptide analogues, was successfully developed and characterized.graphic file with name d6na00729e-ga.webp

1. Introduction

HER2 is a member of the human epidermal growth factor receptor (HER) family and is unique among its members because it lacks a known ligand. Nevertheless, it functions as a preferred co-receptor, forming both homodimers and heterodimers with the other HER family proteins (HER1, HER3, and HER4).1 HER2 gene amplification and/or protein overexpression occur in approximately 20–25% of invasive breast cancers, defining the HER2-positive subtype.2–4 This subtype is associated with poorer prognosis, increased mortality in early-stage disease, a higher incidence of metastasis, and shorter relapse-free survival compared with HER2-negative tumours.5,6 Consequently, HER2 has become a validated therapeutic target in breast cancer, leading to the development of several monoclonal antibody therapies directed against the extracellular domain (ECD) of HER2.7

In this context, the visualization and quantification of drug delivery to the target site are essential for the personalization of therapy.8 Furthermore, active targeting strategies are crucial for minimizing off-target effects and improving therapeutic efficacy.9

Currently, HER2 status determination relies on invasive tissue sampling followed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). However, these methods are influenced by biopsy quality, tumor heterogeneity, and pathological interpretation.

In recent years an increasing interest has been focused on the identification of imaging biomarker and on the development of non-invasive diagnostic tools for cancer characterization and monitoring, which could help in patients' stratification, outcome prediction, and treatment personalization. In this context, non-invasive imaging approaches, besides their noninvasiveness, may capture entire tumor microstructure and heterogeneity, complementing conventional pathological assessment and reducing possible inaccuracy due to sampling errors.

MRI is increasingly used in the evaluation of breast cancer with positive impact on patient management, providing critical information for visualization of complex disease patterns, accurate staging, treatment planning and treatment response monitoring. Recently, a published study also evaluated the potential value of quantitative MRI mapping parameters for determining human HER2 status in breast cancer.10

However, despite its advantages, MRI lacks the molecular specificity provided by direct tissue-based assays.

To circumvent this limitation, liposomal nanocarriers are particularly attractive, since their lipid bilayer provides a versatile platform for the incorporation of functional molecules, including imaging probes and targeting ligands, while their physicochemical properties can be tuned to modulate their biological behaviour.11

To address these challenges, we therefore investigated phospholipid-based nanosystems, specifically liposomes, incorporating magnetic resonance imaging (MRI) contrast agents (CAs).12–15

The incorporation of paramagnetic Gd(iii) complexes into liposomal membranes is an approach that has been extensively investigated over the past decades as a strategy to increase the local concentration of paramagnetic centres and improve the MRI response of the resulting nanostructures.16 In particular, amphiphilic Gd(iii) complexes bearing lipophilic moieties have been incorporated into phospholipid bilayers, and their relaxometric properties have been systematically investigated by several groups.17–21 These studies demonstrated that the relaxometric behaviour of Gd-containing liposomes is strongly influenced by the molecular structure and membrane localization of the paramagnetic complex, as well as by water exchange and the rotational dynamics of both the complex and the liposomal assembly. More recent investigations have further explored the potential of these systems for MRI applications, including in vivo imaging, highlighting the potential of molecularly engineered Gd(iii)-containing amphiphilic systems to modulate MRI contrast efficiency.22 Moreover, other supramolecular soft materials are receiving increasing attention as versatile platforms for the incorporation and delivery of MRI-CAs.23,24

Building on this established approach, the MRI probe developed in the present study is based on an amphiphilic Gd(iii) complex that can be embedded within the phospholipid bilayer, while the metal complex protrudes toward the aqueous environment.25,26 This design allows each liposome to carry multiple copies of the CA, thereby increasing the local concentration of paramagnetic centres and potentially enhancing the detectability of the nanosystem by MRI.

This strategy may also help mitigate some limitations associated with conventional gadolinium-based CAs (GBCAs). Safety concerns have been raised, particularly for certain linear GBCAs, owing to their lower chelate stability and the potential release of free gadolinium ions. Although adverse reactions are uncommon in individuals with normal renal function, the risk of nephrogenic systemic fibrosis (NSF), especially following exposure to less stable linear CAs in patients with severe renal impairment, remains an important clinical concern. Additionally, conventional Gd-based chelates are rapidly cleared from the bloodstream, limiting the available imaging window and reducing their effectiveness for monitoring dynamic biological processes or detecting small pathological lesions. This rapid elimination may reduce sensitivity for the detection of small lesions and impair the visualization of pathological tissues that require prolonged contrast-agent accumulation, such as inflamed atherosclerotic plaques or early metastatic deposits.

Liposomes represent attractive nanocarriers due to their biocompatibility and structural versatility. They consist of phospholipids arranged in one or more bilayers surrounding an aqueous core, enabling the simultaneous encapsulation of both hydrophilic and hydrophobic molecules. Their lipidic nature facilitates efficient uptake by tissues and cells through multiple biological pathways, making them highly suitable for diagnostic and therapeutic applications. In addition, the ability to combine membrane-embedded imaging agents with surface-exposed targeting molecules provides a modular platform in which imaging and molecular recognition can be integrated within the same nanosystem.

To achieve active targeting of HER2-positive tumours, the external surface of the liposomes can be functionalized with a ligand able to target this receptor. A9 (QDVNTAVAW-NH2) is a nine-residue peptide, amidated at the C-terminus, which specifically binds the HER2 receptor and represents a promising alternative to monoclonal antibodies or their fragments for HER2-targeted imaging and therapy.27–30 Although its affinity could be further improved and its hydrophobicity may contribute to nonspecific binding and limited solubility, these drawbacks can be mitigated through liposomal conjugation. Indeed, the exposure of multiple copies of A9 on the liposome surface can enhance binding avidity through multivalent interactions, while the liposomal carrier improves the overall aqueous solubility and the circulation properties of the peptide ligand. In this regard, it is worth mentioning a previously studied ligand-targeted nanoliposome that combines imaging-based diagnosis with targeted chemotherapy. Recognition of the HER2 receptor is achieved through an anti-HER2 peptide identified by screening a phage display peptide library.31

The A9 sequence or its analogues have been conjugated to the liposomal membrane through a polyethylene glycol (PEG) spacer, which enhances ligand accessibility and minimizes steric hindrance that could otherwise reduce binding affinity toward the HER2 receptor. Additionally, the presence of PEG conjugated to a lipid anchor, is reported to significantly enhance the circulation time of liposomes. This targeted design is expected to improve selective accumulation at the tumour site while simultaneously enabling MRI-based visualization and quantification of nanoparticles delivery.

Herein, we report the development of a liposome-based delivery system bearing a GBCA and A9 analogues, HER2-targeting peptides anchored to the liposomal bilayer surface. The efficiency of the formulation strategy was demonstrated through extensive physicochemical characterization of the resulting liposomal system (Dynamic Light Scattering, Nanoparticle Tracking Analysis, Scanning Electron Microscopy, and Low-Field Nuclear Magnetic Resonance). Target recognition and binding were evaluated by flow cytometry (FACS) technique, confirming the ability of the functionalized liposomes to selectively engage HER2-expressing cells. Furthermore, preliminary in vivo proof-of-concept studies were conducted in HER2+ breast cancer xenograft murine model to assess the capability of the GBCA loaded liposomes to distinguish tumour tissue from surrounding healthy tissue. The results demonstrate the potential of this targeted liposomal platform for HER2-directed molecular imaging applications.

2. Results and discussion

2.1. Peptide synthesis

The peptide sequence A10, Cys-A9, was synthesized according to the previously published procedure (Fig. 1).32 The peptide sequence A12 was designed by introducing two PEG units between the active sequence (QDVNTAVAW) and the cysteine residue. These PEG units were added to the N-terminus of A9 to anchor the peptide ligand to the liposome surface. This modification was intended to increase both the distance from the liposome surface and the flexibility of the targeting ligand.

Fig. 1. Chemical structure of peptides A10 and A12.

Fig. 1

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2.2. Preparation and characterization of liposomal formulation

Liposomes are lipid-bilayer vesicles primarily composed of amphiphilic molecules, mainly phospholipids and cholesterol. The specific ratio of the different liposomal components was chosen following the previously published protocol.33 Liposomes were prepared using the thin-film hydration method, followed by extrusion. This method consists of dissolving the individual lipidic component in an organic solvent (chloroform/methanol); the solvent is then evaporated to create a dry lipid film. Through a subsequent hydration of the film with an aqueous phosphate buffer, vesicles can be formed: the obtained dispersion is sonicated and extruded to prepare homogeneous small liposome populations. In Fig. 2 and 3 are shown the schematic view and the formulation composition of developed multifunctional liposomes.

Fig. 2. Schematic representation of Lipo-Gd-Mal: PC/Chol/DSPE-PEG2000-Mal/DSPE-DTPA(Gd) (42/35/3/20).

Fig. 2

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Fig. 3. Schematic representation of Lipo-Gd-Mal-Rhod: PC/Chol/DSPE-PEG2000-Mal/DSPE-DTPA(Gd)/Rhod (42/34/3/20/1).

Fig. 3

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Two formulations were developed, one suitable for MRI studies (Fig. 2), and another one, labeled with a fluorescent dye, suitable to assess the in vitro affinity (Fig. 3) by using a flow cytometry technique (FACS).

To achieve active targeting of the formulated liposomes, A9 peptide sequences were conjugated via Michael addition. This strategy enables site-specific attachment of targeting peptides to the liposomal surface through the formation of a stable covalent thioether bond. For this purpose, a commercial, maleimide-functionalized PEGylated lipid, DSPE-PEG2000-maleimide, was incorporated into the liposomal bilayer. The conjugation reaction was carried out after liposome assembly (post-insertion approach), thereby ensuring greater formulation flexibility and precise functionalization for active targeting. Specifically, the thiol group of the cysteine residue introduced at the N-terminus of the targeting peptide sequence (Scheme 1) reacted with the maleimide moieties under slightly basic aqueous conditions, resulting in efficient coupling of the targeting ligand to the liposome surface.

Scheme 1. Peptide (A10, A12) conjugation on liposomal surface: Lipo-Gd-Mal-PEPTIDE (A), Lipo-Gd-Mal-Rhod-PEPTIDE(B).

Scheme 1

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In detail, liposomes were prepared in 100 mM PBS buffer at a final lipid concentration of 5 mM. Selected formulations were subsequently functionalized with the targeting peptides A10 and A12 for further characterization. The efficiency of peptide conjugation was 91.5% for Lipo-Gd-Mal-A10 and 92.3% for Lipo-Gd-Mal-A12, as determined by quantification of the unbound peptide fraction recovered after gel filtration using UV-Vis analysis. These values allowed the calculation of peptide-to-total-lipid molar ratios of 0.0128 and 0.0129 for A10- and A12- functionalized liposomes, respectively. A mean particle diameter below 200 nm and a narrow size distribution (PDI ≤ 0.2) were selected as design criteria, as these physicochemical characteristics are widely recognized as favorable for the development of liposomal nanocarriers intended for biological applications.34

Specifically, following preparation, the formulations were characterized by Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), Scanning Electron Microscopy (SEM), and Low-Field Nuclear Magnetic Resonance (LF-NMR).

2.2.1. DLS analysis

By employing the DLS technique, the presence of nanosized particles in all formulations, with a mean Z-average diameter of 95.6 ± 10.2 nm was revealed (Fig. 4 and Table 1).

Fig. 4. DLS intensity profiles of: (A) Lipo-Gd-Mal; (B) Lipo-Gd-Mal-A10; and (C) Lipo-Gd-Mal-A12. Solid lines represent freshly prepared formulations (t = 0), whereas dashed lines represent the corresponding formulations after 30 days of storage.

Fig. 4

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Table 1. DLS characterization of the liposomal formulations: hydrodynamic diameter (Dh), polydispersity index (PDI), and zeta potential (ζ). Values are reported as mean ± standard deviation (S.D.).
Formulation D h (nm) ± S.D. PDI ± S.D. ζ (mV) ± S.D.
graphic file with name d6na00729e-u1.jpg 95.6 ± 10.2 0.20 ± 0.02 −2.89 ± 0.32
graphic file with name d6na00729e-u2.jpg 96.5 ± 8.5 0.15 ± 0.01 −7.66 ± 0.34
graphic file with name d6na00729e-u3.jpg 95.4 ± 11.2 0.14 ± 0.02 −9.82 ± 0.43
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Peptide functionalization did not result in appreciable changes in particle size, as comparable diameters were observed for both non-functionalized and peptide-modified liposomes (Table 1). Consistently, all formulations exhibited low PDI values, ranging from 0.14 to 0.20, suggesting the formation of relatively homogeneous nanoscale populations. Moreover, no significant variations in particle size were observed after 30 days, indicating good colloidal stability of the liposomal formulations over time (Fig. 4).

2.2.2. NTA analysis

To further evaluate particle size distribution and determine particle concentration, the liposomal formulations were analyzed by NTA. As shown in Fig. 5, the main particle population was centered around 100 nm regardless of surface functionalization. Mean particle diameters determined by NTA ranged from 98.7 ± 2.1 nm for Lipo-Gd-Mal-A10 to 108.8 ± 2.8 nm for the non-peptide-functionalized liposomal formulation, in agreement with the values obtained by DLS (Table 2). The reported NTA values represent the mean particle diameter ± standard error (S.E.) obtained from five video acquisitions and therefore describe the uncertainty of the estimated mean rather than the overall particle-size distribution. Although DLS and NTA rely on different analytical principles, both techniques provided comparable mean particle-size values in the nanoscale range. These findings suggest that peptide conjugation did not substantially affect the mean vesicle dimensions.

Fig. 5. Size distribution of particles using NTA for (A) Lipo-Gd-Mal; (B) Lipo-Gd-Mal-A10 functionalized with A10 peptide; and (C) Lipo-Gd-Mal-A12 functionalized with A12 peptide.

Fig. 5

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Table 2. NTA characterization of the liposomal formulations: mean hydrodynamic diameter (Dh), standard deviation (S.D.) of the particle-size distribution, and particle concentration. Values are reported as mean ± standard error (S.E.).
Formulation D h (nm) ± S.E. S.D. ± S.E. Concentration ± S.E. × 1014 (number of particles per mL)
graphic file with name d6na00729e-u4.jpg 108.8 ± 2.8 51.6 ± 1.0 1.45 ± 0.13
graphic file with name d6na00729e-u5.jpg 98.7 ± 2.1 49.3 ± 2.2 1.88 ± 0.18
graphic file with name d6na00729e-u6.jpg 107.4 ± 0.9 59.7 ± 1.1 2.43 ± 0.15
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NTA analysis also enabled the estimation of particles concentration, yielding values on the order of 1014 particles per mL for all formulations. Specifically, nanoparticle concentrations increased from 1.45 × 1014 ± 1.3 × 1013 particles per mL for non-peptide-functionalized liposomes to 1.88 × 1014 ± 1.8 × 1013 and 2.43 × 1014 ± 1.5 × 1013 particles per mL for A10- and A12-functionalized liposomes, respectively. Despite these differences, all formulations maintained comparable mean particle sizes and low dispersity, indicating that peptide functionalization did not substantially alter the overall physicochemical characteristics of the liposomal systems.

Zeta-potential measurements revealed a negative surface charge for all formulations, with values shifting from −2.89 mV for non-functionalized liposomes to −7.66 mV and −9.82 mV following functionalization with A10 and A12, respectively. The observed decrease in zeta-potential is consistent with changes occurring at the liposomal surface after peptide conjugation and suggests successful surface modification. The progressive increase in negative surface charge following functionalization may reflect the contribution of peptide-associated ionizable groups exposed at the liposomal interface (Table 1).

Overall, physicochemical characterization indicated that all formulations met the predefined design criteria, exhibiting nanoscale dimensions and reproducible colloidal properties suitable for subsequent biological evaluation.

2.2.3. SEM analysis

Liposome size, morphology, and surface characteristics were further investigated by SEM. Representative micrographs (Fig. 6) revealed predominantly spherical vesicles with diameters below 100 nm, in agreement with the particle size measurements obtained by DLS and NTA. In addition to individual particles, the images showed the presence of particle aggregates, which were likely generated during sample deposition, air-drying, and sputter-coating procedures prior to SEM analysis. It should also be considered that SEM analysis is performed on dehydrated samples under vacuum conditions; therefore, the observed size and morphology may not fully reflect those of liposomes in their native hydrated state, as sample preparation can induce structural alterations and dimensional changes.

Fig. 6. SEM micrographs (left panels) of: (A) Lipo-Gd-Mal; (B) Lipo-Gd-Mal-A10; and (C) Lipo-Gd-Mal-A12. Corresponding elemental maps (right panels) obtained via Energy-Dispersive X-ray Spectroscopy (EDS) using ChemiSEM technology, showing the distribution of gadolinium (Gd, highlighted in orange) within the liposomal structures. Scale bars: 4 µm for all images.

Fig. 6

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The spherical morphology observed by SEM is consistent with the structural organization typically reported for phospholipid-based nanoparticles.35 Moreover, the agreement between SEM, DLS, and NTA measurements supports the formation of reproducible nanosized liposomal populations and indicates that peptide functionalization did not result in appreciable morphological alterations.

Overall, SEM observations were consistent with the formation of nanosized liposomal structures and provided complementary morphological information to the results obtained from light-scattering techniques.

Elemental analysis was performed using ChemiSEM technology, which integrates SEM imaging with energy-dispersive X-ray spectroscopy (EDS). Elemental mapping revealed the presence of gadolinium-associated signals within the analyzed liposomal samples. The spatial distribution of the metal signal overlapped with the liposomal structures observed in the SEM micrographs, supporting the presence of GBCAs within the liposomal formulation. These findings are consistent with the successful preparation of Gd-containing liposomes and further corroborate the incorporation of the gadolinium-functionalized lipid during liposome assembly.

2.2.4. LF-NMR characterization

Low-field nuclear magnetic resonance (LF-NMR) relaxivity measurements were performed at a proton Larmor frequency of 20 MHz and at four gadolinium concentrations (0.1, 0.5, 0.8, and 1.0 mM) showed that all liposomal formulations exhibited higher longitudinal relaxation rates (R1) compared with aqueous Gd-DTPA solutions at equivalent gadolinium concentrations (Fig. 7A).

Fig. 7. A) Longitudinal relaxation rate (R1) measurements of aqueous Gd-DTPA solutions at 0.1, 0.5, 0.8, and 1.0 mM (black circles), and of the corresponding Lipo-Gd-Mal formulations containing equivalent Gd complex concentrations: non-functionalized liposomes (green triangles) and peptide-functionalized liposomes with A10 (red squares) and A12 (blue diamonds) peptides. (B) Relaxivity values (mM−1 s−1) of Gd-DTPA aqueous solution and of Lipo-Gd formulations at 20 MHz, 310 K.

Fig. 7

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The corresponding relaxivity values (r1) were determined from the slope of the linear regression of the R1 as a function of gadolinium concentration (Fig. 7B).

The increase in relaxation rates observed for the Gd-liposomal systems is expected because it is well-known that the incorporation of gadolinium complexes within the liposomal bilayer can influence the CA.36,37 rotational correlation time τR, strictly related to the size and to the rigidity of the investigated system. Peptide-functionalized liposomes (A10 and A12) showed R1 values comparable to those of non-functionalized liposomes, indicating that surface modification and the presence of the ethoxylic spacer in A12 did not substantially affect the relaxometric behavior of the system.38

Liposomal formulations exhibited r1 values of 9.36 ± 0.18 mM−1 s−1 (Lipo-Gd), 8.93 ± 0.17 mM−1 s−1 (Lipo-Gd-Mal-A10), and 8.81 ± 0.16 mM−1 s−1 (Lipo-Gd-Mal-A12), whereas the aqueous Gd-DTPA solution showed an r1 value of 3.73 ± 0.16 mM−1 s−1. The relaxivity value obtained for Gd-DTPA is consistent with previously reported data acquired under comparable magnetic field strength and temperature conditions.39,40

Overall, the increase in relaxivity observed for liposomal formulations compared with free Gd-DTPA is consistent with reduced rotational mobility of the gadolinium-containing lipid once incorporated into the liposomal bilayer, a factor known to contribute to enhanced r1 values in nanoparticulate contrast agents.41 The absence of significant differences between functionalized and non-functionalized liposomes further suggests that peptide conjugation does not interfere with the relaxometric properties of the system.

Although relaxivity values were obtained at a single magnetic field strength (20 MHz, 0.47 T), the observed trends are consistent with typical behavior reported for lipid-based nanosystems at low magnetic fields.21,42–44 These findings support the potential of the developed liposomal platforms for further evaluation as MRI contrast agents under relevant preclinical conditions.

2.3. In vitro study

2.3.1. Cytotoxicity

The cytotoxicity of the liposomal formulations was evaluated in BT-474 cells after 48 h of incubation by the MTS assay. As shown in Fig. 8, cell viability in samples treated with Lipo-Gd remained essentially unchanged compared to the untreated control cells, indicating that the gadolinium-containing formulation did not exhibit significant cytotoxic effects.

Fig. 8. Cell viability of BT-474 cells. Cells were treated for 48 h with Lipo-Gd-Mal, Lipo-Gd-Mal-A10, or Lipo-Gd-Mal-A12, cell viability was evaluated by MTS assay. Results are expressed as percentage of viable cells relative to untreated control cells (CTR) (*p < 0.05; **p < 0.001; ***p < 0.0001).

Fig. 8

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Similarly, treatment with the peptide-functionalized liposomal systems did not reduce cell viability; on the contrary, both Lipo-Gd-Mal-A10 and Lipo-Gd-Mal-A12 were associated with higher viability values than untreated cells, confirming the good biocompatibility of the functionalized formulations. Overall, all the tested formulations were well tolerated by BT-474 cells at the concentrations used, with the A10-functionalized liposomes showing a slightly more favorable viability profile than the A12-functionalized formulation, suggesting that it may represent the most promising system for further investigation.45–48

2.3.2. Binding experiment

The binding ability of the two functionalized liposomal systems, Lipo-Gd-Mal-A10-Rhod and Lipo-Gd-Mal-A12-Rhod, to HER2-overexpressing BT-474 cells was evaluated by flow cytometry (FACS) and compared with that of non-functionalized liposomes used as a control (Fig. 9 panel A).

Fig. 9. Fluorescence-activated cell sorter (FACS) profiles of BT-474 (panel A) and for BT-549 cell lines (panel B), treated with Lipo-Gd-Mal-A10-Rhod, Lipo-Gd-Mal-A12-Rhod and Lipo not functionalized with peptide, as control, at final liposomal concentration of 0.2 mM (1% rhodamine B); median fluorescence intensity (MFI) of the gated fluorescent-positive cell population in BT-474 and BT-549 cells (panel C) (**p < 0.001; ***p < 0.0001).

Fig. 9

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As shown in the figure, the liposomes functionalized with peptide A10 exhibited the highest binding affinity, reaching 54.9%, whereas the formulation bearing the A12 peptide on the liposomal surface showed a slightly lower, yet still substantial, binding affinity (44.7%). Importantly, both functionalized formulations markedly increased cell binding compared with non-functionalized liposomes, which bound to only 20.2% of HER2-positive cells (Fig. 9 panel A). These results indicate that the selected targeting peptides significantly enhance the recognition of BT-474 cells through a selective interaction with the HER2 receptor, consistent with the high expression of the HER2 receptor on the surface of this cell line.

To further investigate the selectivity of the peptide-functionalized liposomes toward the HER2 receptor, the same FACS analysis was performed on BT-549 cells, which exhibit low levels of HER2 expression (Fig. 9 panel B).

In contrast to the results obtained with BT-474 cells, only a weak fluorescence signal was detected in BT-549 cells, with no significant increase in the percentage of fluorescent for both peptide-functionalized liposomes. This finding is consistent with the specific interaction between the HER2 receptor and the peptide ligands displayed on the liposome surface. Overall, these results support the involvement of HER2-mediated interactions in liposome binding and provide further evidence for the target specificity of the developed liposomal systems as an additional quantitative assessment, the median fluorescence intensity (MFI) of the gated fluorescent-positive cell population was analyzed (Fig. 9 panel C). In BT-474 cells, A10- and A12-functionalized liposomes showed 3.6- and 3.9-fold higher MFI, respectively, than non-functionalized liposomes. In contrast, only marginal increases were observed in BT-549 cells (1.07-fold for A10 and 1.05-fold for A12). These findings further support the HER2-dependent cellular binding of the peptide-functionalized liposomes.

Among the two formulations, the A10-functionalized liposomes exhibited the greatest binding efficiency, suggesting that this peptide sequence represents the most promising candidate for further in vivo investigation targeting and biodistribution studies.49

2.4. In vivo study

Following subcutaneous injection of BT-474 HER2-positive breast cancer cells in athymic NU/NU female mice, tumour engraftment was observed with High Frequency Ultrasound (HFUS) imaging, in 3 out of 5 mice after about 3 months, allowing the xenograft model to be used for the subsequent in vivo MRI proof-of-concept study. At the time of MRI examination, all tumours were detectable on T1-weighted images. The 3 mice were randomly assigned, in a blinded manner, to receive an intravenous injection of 150 µL of free Gd-DTPA, Lipo-Gd-Mal, or Lipo-Gd-Mal-A10, respectively. This preliminary experiment was used to evaluate the potential of the A10 peptide-functionalized liposomal system as an MRI CA compared with the non-functionalized formulation and free Gd-DTPA. All administered formulations contained the same nominal gadolinium concentration (20 mM), which, given the injected volumes, corresponded to a total dose of 3 µmol of Gd per mouse, allowing for a meaningful comparison among the formulation.

The MR images were acquired before and at multiple time points after injection (Fig. 10). For each time point, the tumour signal was measured and normalised to the signal intensity of an adjacent muscle. The raw normalized signal values measured for each CA used were reported in Table 3. Enhancement profiles of the CAs were evaluated to investigate differences in tumour accumulation and retention over time (Fig. 11).

Fig. 10. In vivo MR images acquired with T1-weighted RARE sequences in the three mice, respectively injected with free Gd-DTPA, Lipo-Gd-Mal, or Lipo-Gd-Mal-A10. The reported images are acquired before contrast agent administration (first column) and at 0 and 180 min after injection. In the pre-contrast image also the ROI tumour contoured in red is reported.

Fig. 10

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Table 3. Normalized tumor signal values measured for each of the three mice undergoing MRI examination, each receiving a different contrast agent (Gd-DTPA, Lipo-Gd-Mal-A10, Lipo-Gd-Mal). Values were measured before contrast agent administration and at 0, 10 min, 20 min, 30 min, 1 h, 2 h and 3 h after injection.

Pre-contrast 0 min 10 min 20 min 30 min 60 min 120 min 180 min
Gd-DTPA 1014 1373 1321 1269 1259 1195 1110 1040
graphic file with name d6na00729e-u7.jpg 1094 1136 1223 1163 1014 1091 1361 1276
graphic file with name d6na00729e-u8.jpg 1037 1146 1143 1129 1024 1126 0996 1006
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Fig. 11. In vivo MRI assessment of tumour signal enhancement following intravenous injection of the different formulations. Time-dependent tumour signal enhancement profiles following injection of free Gd-DTPA, non-functionalized Gd-DTPA-loaded liposomes (Lipo-Gd-Mal), and A10 peptide-functionalized Gd-DTPA-loaded liposomes (Lipo-Gd-Mal-A10). Signal enhancement was calculated at multiple time points after CA administration to compare the tumour accumulation and retention kinetics of the different formulations.

Fig. 11

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In the mouse injected with Gd-DTPA, tumour enhancement reached its maximum immediately after administration. In the mouse receiving Lipo-Gd-Mal-A10, tumour enhancement reached a level comparable to that observed with free Gd-DTPA approximately 2 h after the injection. This delayed enhancement profile may be consistent with the prolonged biodistribution of the liposomal formulation.

In the mouse injected with control Lipo-Gd-Mal, only limited tumour enhancement was observed throughout the observation period. These findings provide preliminary observations suggesting that A10 peptide functionalization may contribute to tumour accumulation of Gd-DTPA-loaded liposomes. Overall, these results indicate the feasibility of using this targeted nanoplatform as a molecular MRI CA for HER2-positive breast cancer imaging and warrant further investigation in studies with larger sample size.

3. Materials and methods

3.1. Materials

Fmoc-protected amino acids, rink amide MBHA resin, N-hydroxybenzotriazole (HOBt) and benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium (PyBOP) and all solvents were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA); piperidine and diisopropylethylamine (DIPEA) were purchased from Fluka (Milwaukee, WI, USA); SiliaBond DMAP was purchased from Silicycle (Québec, QC, G1P 4S6, Canada) diethylenetriaminepentaacetic acid gadolinium(iii) dihydrogen salt hydrate (Gd-DTPA) was acquired from Sigma-Aldrich, (Bucks, Switzerland). l-α-Phosphatidylcholine (95%) (PC), cholesterol from sheep wool (≥92.5%) (Chol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] ammonium salt (DSPE-PEG2000-Maleimide), 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (gadolinium salt) (DSPE-DTPA(Gd)), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt (Rhod PE) were purchased from Avanti Research™ (Alabaster, AL, USA). Phosphate-buffered saline (PBS, 100 mM, pH 7.4) was prepared by dissolving PBS tablets (P4417, Sigma-Aldrich) in ultrapure water according to the manufacturer's instructions. The resulting buffer contained sodium chloride (137 mM), potassium chloride (2.7 mM), and phosphate salts (100 mM sodium/potassium phosphate buffer).

3.2. Preparation of liposomal formulations

Three liposomal formulations were prepared:

✓ Maleimide-functionalized liposomes (Lipo-Mal), PC/Chol/DSPE-PEG2000-Mal (52/45/3 molar ratio).

✓ Gadolinium-derivatized liposomes (Lipo-Gd-Mal), PC/Chol/DSPE-PEG2000-Mal/DSPE-DTPA(Gd) (42/35/3/20 molar ratio).

✓ Dual-labeled gadolinium/rhodamine liposomes (Lipo-Gd-Mal/Rhod), PC/Chol/DSPE-PEG2000-Mal/DSPE-DTPA(Gd)/Rhod PE (42/34/3/20/1 molar ratio).

All liposomal formulations were prepared by the thin-film hydration method.50 Briefly, all components were dissolved in chloroform, whereas DSPE-DTPA(Gd) was dissolved in a chloroform/methanol mixture (1 : 1, v/v). The lipid stock solutions were combined at the desired molar ratios, and the organic solvents were evaporated under a gentle nitrogen stream to form a homogeneous lipid film. The resulting film was hydrated with 1.0 mL of 100 mM PBS (pH 7.4) to obtain a final lipid concentration of 5 mM. The suspension was sonicated for 30 min and subsequently extruded 10 times at room temperature using a ThermoBarrel Extruder system (Northern Lipids Inc., Vancouver, BC, Canada) under a nitrogen atmosphere through a 0.1 µm polycarbonate membrane (Nucleopore Track Membrane, 25 mm; Whatman, Brentford, UK). For peptide functionalization, 35 µL of peptide solution (1 mM in ultrapure water) was added to 500 µL of maleimide-containing liposomes. The reaction mixture was stirred at room temperature overnight, allowing covalent coupling between the thiol groups of the peptide and the maleimide moieties exposed on the liposomal surface. Unconjugated peptide was subsequently removed by gel filtration on a pre-packed Sephadex G-50 column prior to further characterization and biological studies. The conjugation efficiency was calculated from the amount of unbound peptide recovered after gel filtration and quantified by UV-Vis analysis. The peptide-to-total-lipid molar ratio was calculated as the molar amount of conjugated peptide divided by the total molar amount of lipids in the formulation.

3.3. Liposomal characterization

3.3.1. Dynamic light scattering (DLS) and ζ-potential measurements

The hydrodynamic diameter (Dh), polydispersity index (PDI), and ζ-potential of liposomes were determined using a Zetasizer Ultra instrument (Malvern Instruments, Worcestershire, UK). Measurements were performed at 25 °C using a backscatter detection angle of 173° and disposable polystyrene cuvettes. Prior to analysis, liposomal suspensions were centrifuged at 13 000 rpm for 5 min at room temperature to remove potential aggregates. All measurements were carried out in triplicate, and data are reported as mean ± standard deviation (SD). ζ-Potential measurements were performed after appropriate dilution of samples in Milli-Q water to minimize the effects of ionic strength.

3.3.2. Nanoparticles tracking analysis (NTA) measurements

Particle size distribution and particle concentration were further evaluated by nanoparticle tracking analysis (NTA) using a NanoSight NS300 instrument (Malvern Panalytical, UK). Liposome suspensions were diluted in double-distilled water to achieve a particle concentration within the optimal measurement range of 20–100 particles per frame. Five independent videos were acquired for each sample and analyzed using the NanoSight software under standard measurements conditions. The mean particle diameter and associated S.E. were obtained directly from the software output.

3.3.3. Scanning electron microscopy (SEM)

Morphology of liposomes was examined using a field-emission scanning electron microscope (Phenom Pro X G6, Thermo Fisher Scientific, Eindhoven, The Netherlands). Liposomal suspensions were diluted 1 : 1000 in ultrapure water, and 20 µL of each sample was deposited onto aluminum stubs covered with conductive carbon adhesive tape. Samples were air-dried at room temperature and sputter-coated with a 7 nm gold layer at 20 mA for 60 s. Imaging was performed at an accelerating voltage of 15 kV under high-vacuum conditions using a secondary electron detector. Elemental composition was analyzed by energy-dispersive X-ray spectroscopy (EDS) integrated within the ChemiSEM system.

3.3.4. Low-field nuclear magnetic resonance (LF-NMR)

Low-field nuclear magnetic resonance (LF-NMR) measurements were performed at 310 K using a Bruker Minispec mq20 spectrometer (0.47 T, 20 MHz; Bruker, Germany). Prior to analysis, samples were equilibrated at 310 K for approximately 15 min by placement within the probe to ensure thermal stability.

Experiments were carried out using aqueous Gd-DTPA solutions in the concentration range of 0.1–1.0 mM and Gd-loaded liposome dispersions containing equivalent gadolinium concentrations. Aqueous Gd-DTPA solutions were prepared by serial dilution of a stock solution in Milli-Q water, whereas Gd-loaded liposomes were diluted from the corresponding stock formulation in Milli-Q water. The reported concentrations refer to the final gadolinium concentration in each sample. Longitudinal relaxation times (T1) were determined using an inversion recovery pulse sequence and fitted with a multi-exponential model where appropriate. The longitudinal relaxation rate (R1) was calculated as the reciprocal of T1 (R1 = 1/T1) and relaxivity values (r1) were obtained as the slope of the linear regression of the R1versus gadolinium concentration using a least-squares fitting approach.

3.4. In vitro study

The human breast cancer cell line BT-474 was provided by the Cell Culture Facility of CEINGE-Biotecnologie Avanzate “Franco Salvatore” (Naples, Italy). The BT-549 cells were purchased and authenticated by the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured in RPMI medium supplemented with 20% and 10% fetal bovine serum (FBS), respectively, and a 1% solution of l-glutamine, penicillin, and streptomycin and maintained at 37 °C with 5% CO2. BT 474 cells are characterized by ERBB2/HER2 gene amplification, resulting in high HER2 surface expression and constitutive activation of downstream signaling pathways. This HER2 positive phenotype is routinely used as a model for HER2 driven breast cancer biology and for evaluating HER2 targeted interventions.

3.4.1. Cell viability assay (MTS assay)

BT-474 cells were seeded in 96-well plates at a density of 5 × 103 cells per well and allowed to adhere for 24 h under standard culture conditions. Cells were treated with liposomes alone or with liposome–peptide complexes (A12-Gd or A10-Gd) at final concentrations of 0.2 mM liposomes, 0.04 mM Gd for 48 h. Cell viability was evaluated using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega BioSciences Inc., Fitchburg, WI, USA), based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), according to the manufacturer's instructions. At the end of each treatment, the reagent was added to each well and the plates were incubated for the recommended time. Absorbance was then measured using a microplate reader, and cell viability was expressed as percentage relative to untreated control cells. The final values represent the mean ± standard deviation of three independent experiments. Statistical significance was assessed using Student's t-test, with p < 0.05 considered significant.

3.4.2. Binding assay by FACS analysis

BT-474 and BT-549 cells were seeded in 6-well plates at a density of 3.5 × 105 cells per well. After 24 hours, cells were incubated for 1 hour at 37 °C in the dark with liposomes alone or with liposome–peptide complexes (A12-Gd-Rho or A10-Gd-Rho) at final concentrations of 0.2 mM liposomes, 0.04 mM Gd, and 1% rhodamine B. Rhodamine B was chosen for its emission spectrum, which minimizes overlap with cellular autofluorescence. After incubation, the cells were detached using trypsin and centrifuged at 1500 rpm for 5 minutes. The pellets were washed twice with PBS to remove unbound complexes and then resuspended in 500 µL of PBS. The samples were analyzed on a CytoFLEX flow cytometer (Beckman Coulter), data analysis was performed using CytExpert software (Beckman Coulter). The final values represent specific binding levels obtained from three independent incubations and are expressed as mean ± standard deviation. Statistical significance was assessed using Student's t-test, with p < 0.05 considered significant. The median fluorescence intensity (MFI) of the gated fluorescent-positive cell population was evaluated.

3.5. In vivo study

All animal experiments were conducted in accordance with Italian and European regulations governing the use of animals for scientific purposes and with the FELASA guidelines. The study was approved by the Italian Ministry of Health (Authorization No. 843/2024-PR) and carried out at CEINGE – Biotecnologie Avanzate Franco Salvatore Scarl (Naples, Italy) under the institutional protocol (Ministry Decree No. 19/2024-UT). 5 Six-week-old female athymic NU/NU mice (18–20 g) were purchased from Charles River Laboratories (Milan, Italy) and were allowed to acclimatize to the animal facility for one week before the start of the experimental procedures. Animals were housed in individually ventilated cages under controlled environmental conditions (22 ± 2 °C, 55 ± 10% relative humidity) with a 12 h light/dark cycle and were provided with standard rodent chow and irradiated water ad libitum.

All the in vivo procedures were performed under general anesthesia induced with 5% isoflurane in oxygen (0.8 L min−1) in an induction chamber and maintained with 1.5–3% isoflurane delivered through a nose cone, with the anesthetic concentration adjusted according to the duration and invasiveness of the procedure.

3.5.1. HER2+ breast cancer xenograft murine model

BT-474 were cultured in CEINGE-Biotecnologie Avanzate Franco Salvatore Cell Colture Facility in RPMI 1640 medium (cat. n. R0883, Sigma-Merck, Milan, Italy), supplemented with 20% fetal bovine serum (FBS, cat. n. A52566701, Gibco, Fisher Scientific Italia, Milan) and 1% l-Glutamine (cat. n. 25030024, Gibco, Fisher Scientific Italia, Milan). Cells were incubated at 37 °C in a humidified 5% CO2 atmosphere. Cells were routinely tested for mycoplasma (MycoAlert, cod. LT07-318, Lonza Bioscience, Basel, Switzerland).

To establish the tumor model, BT-474 HER2-positive breast cancer cells were inoculated by heterotopic subcutaneous injection into the right flank of the animals. Before injection, cells were resuspended in a final volume of 100 µL consisting of a 1 : 1 mixture of sterile saline and Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). A total of 6 × 106 BT-474 cells were administered in a single subcutaneous injection to generate xenograft tumors.

Tumour development was monitored weekly using a HFUS imaging system (F2 LAZR-X20, FUJIFILM VisualSonics Inc., Toronto, ON, Canada) equipped with a 30–50 MHz MS550D transducer. Once the tumors were visible, the mice underwent MRI.

3.5.2. Magnetic resonance imaging

The xenograft BT-474 tumour-bearing mice were blindly divided into three groups, according to the formulation used as contrast agent, free Gd-DTPA, Lipo-Gd-Mal, Lipo-Gd-Mal-A10, all at a nominal Gd concentration of 20 mM. The experiment was performed on a Bruker BioSpec at 9.4 T. To facilitate venous access, the lateral tail veins were dilated by immersing the tail of the mouse in warm water (38–39 °C) for 3 minutes. A catheter, consisting of a 30-gauge needle connected to polyethylene tubing, was inserted into the vein and secured using surgical glue. The animal was placed onto the MRI bed in decubitus positioning and, after coil centering, the MRI scan started. The acquired protocol consisted in a series of T1-weighted RARE sequences acquired before and after (at 0, 10 min, 20 min, 30 min, 1 h, 2 h and 3 h) the intravenous injection of 150 µL of CA (3 µmol of Gd per mouse). Notably, the same animal positioning was maintained up to 1 h sequence. The last two were performed in separate anesthesia sessions, resulting in different animal positioning between the images. The sequence parameters are: TE = 8.7 ms, TR = 461.5 ms, slice thickness 0.5 mm, acquisition matrix = 256 × 256, pixel size = 0.12 × 0.12, NEX = 3.

3.5.3. Image processing

For each animal, the tumour was identified on the baseline T1-weighted images and the slice showing the largest tumour cross-sectional area was selected for following analysis. A region of interest (ROI) was manually drawn to encompass the entire visible tumour area on the selected slice, while avoiding the surrounding healthy tissue. The same ROI was propagated to all post-contrast images acquired up to 1 h, maintaining identical positioning. For the 2 h and 3 h acquisitions, the ROI was manually adjusted to account for changes in animal positioning. The mean MR signal intensity within the tumour ROI was measured at each time point. To minimize the influence of signal variations between time points and mice, the tumour signal was normalized to the signal intensity measured in a circular reference ROI placed in the adjacent non-tumour tissue. Accordingly, the normalized signal at each time point i was given by Si = ROIt,i/ROIm,i, where ROIt,i and ROIm,i represent the mean signal intensities measured in the tumour and in a reference muscle, respectively. The signal enhancement was then calculated from the normalized values at each time point relative to the corresponding pre-contrast measurement (Spre), according to the following equation:Enhancementi (%) = 100 × (Si − Spre)/Spre

4. Conclusions

A HER2-targeted selective liposomal platform, incorporating an amphiphilic Gd(iii) MRI-CA and externally derivatized with A9 peptide analogues, was successfully developed. This design combines active molecular targeting with MRI signal amplification by exploiting the high payload of CAs incorporated within each liposome. The resulting formulations were comprehensively characterized in terms of size, morphology, and colloidal properties. Moreover, all developed formulations showed good biocompatibility and negligible cytotoxicity, under the employed experimental conditions. Their targeting capability was evaluated by flow cytometry, demonstrating selective recognition of HER2-expressing cells and confirming that the surface-conjugated peptide ligand retained its orientation compatible with an efficient receptor binding.

Furthermore, preliminary proof-of-concept in vivo studies in a HER2+ breast cancer xenograft mouse model showed that the gadolinium-loaded targeted liposomes may enable MRI discrimination of tumour tissue from surrounding healthy tissue.

These preliminary findings also highlight the potential of this multifunctional liposomal platform as an in vivo theranostic delivery system for chemotherapeutic agents, although further studies with larger sample sizes are required to investigate and validate these effects. Indeed, therapeutic drugs can be encapsulated within the aqueous core or incorporated into the lipid bilayer of the liposomes, depending on their solubility or other physicochemical properties. Drug delivery can be monitored non-invasively by MRI through the incorporated gadolinium-based contrast agent, while selective accumulation at the tumour site is promoted by the surface-displayed peptide ligand, which specifically recognizes and binds the HER2 receptor. This integrated theranostic approach combines targeted drug delivery with non-invasive imaging, offering a promising strategy for image-guided cancer therapy and personalized medicine.

Conflicts of interest

There are no conflicts to declare.

Abbreviations

A9

QDVNTAVAW-NH2 peptide

A10

Cys-A9 peptide analogue

A12

PEGylated Cys-A9 peptide analogue

BT-474

Human breast cancer cell line

BT-549

Human breast cancer cell line

HER2

Overexpressing breast cancer cell line

CAs

Contrast agents

Chol

Cholesterol

DLS

Dynamic light scattering

DTPA

Diethylenetriaminepentaacetic acid

DSPE

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine

DSPE-DTPA(Gd)

Gadolinium-containing DSPE-DTPA lipid

DSPE-PEG2000-Mal

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]

ECD

Extracellular domain

EDS

Energy-dispersive X-ray spectroscopy

ERBB2

Erb-B2 receptor tyrosine kinase 2 gene

FACS

Fluorescence-activated cell sorting

FBS

Fetal bovine serum

GBCA

Gadolinium-based contrast agent

Gd(iii)

Gadolinium(iii)

Gd-DTPA

Gadolinium-diethylenetriaminepentaacetic acid

HER

Human epidermal growth factor receptor

HER2

Human epidermal growth factor receptor 2

HFUS

High frequency ultrasound

LF-NMR

Low-field nuclear magnetic resonance

Lipo-Gd

Gadolinium-loaded liposomes

Lipo-Gd-Mal

Maleimide-functionalized gadolinium-loaded liposomes

Lipo-Gd-Mal-Rhod

Gadolinium/rhodamine-labelled maleimide-functionalized liposomes

MRI

Magnetic resonance imaging

NTA

Nanoparticle tracking analysis

NSF

Nephrogenic systemic fibrosis

PBS

Phosphate-buffered saline

PC

Phosphatidylcholine

PEG

Polyethylene glycol

PDI

Polydispersity index

R 1

Longitudinal relaxation rate

r 1

Longitudinal relaxivity

Rhod

Rhodamine

ROI

Region of interest

SEM

Scanning electron microscopy

SD

Standard deviation

T 1

Longitudinal relaxation time

ζ-potential

Zeta potential

Acknowledgments

The authors acknowledge financial support from the Italian Ministry for University and Research (MUR) through the PRIN 2022-PNR project (Grant P2022MPZ3T to S. D. L.). E. G. was supported by the Italian Ministry of Health – Ricerca Corrente Project. Y. F., L. L., C. T., A. S. were supported by the project “StrEngthEning the ItaLIan network of the European research InFrastructure EuroBioImaging”, acronym “SEELIFE” (Project No. IR0000023, CUP B53C22001810006), funded by the European Union's NextGenerationEU under the MUR call 3264/2021 PNRR M4/C2/L3.1.1. The authors thank Euro-BioImaging and the Multi-Modal Molecular Imaging Italian Node Facility at the Institute of Biostructures and Bioimaging (CNR), Naples, for the support with preclinical research. The authors also thank Leopoldo Zona, Luca De Luca, Giorgio Varriale, Massimiliano Mazzucchi, and Maurizio Amendola for the technical assistance.

Data availability

The data supporting the findings of this study include the experimental datasets generated for the physicochemical characterization and biological evaluation of the developed liposomal formulations, comprising Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), Scanning Electron Microscopy (SEM), Low-Field Nuclear Magnetic Resonance (LF-NMR), cell viability (MTS), flow cytometry (FACS), and in vivo MRI experiments. Representative data are presented within the article. Additional raw data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data supporting the findings of this study include the experimental datasets generated for the physicochemical characterization and biological evaluation of the developed liposomal formulations, comprising Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), Scanning Electron Microscopy (SEM), Low-Field Nuclear Magnetic Resonance (LF-NMR), cell viability (MTS), flow cytometry (FACS), and in vivo MRI experiments. Representative data are presented within the article. Additional raw data supporting the findings of this study are available from the corresponding author upon reasonable request.

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