Dual ligand functionalized pH-sensitive liposomes for metastatic breast cancer treatment: in vitro and in vivo assessment

Abstract

This research demonstrates the design and development of a novel dual-targeting, pH-sensitive liposomal (pSL) formulation of 5-Fluorouracil (5-FU), i.e., (5-FU-iRGD-FA-pSL) to manage breast cancer (BC). The motivation to explore this formulation is to overcome the challenges of systemic toxicity and non-specific targeting of 5-FU, a conventional chemotherapeutic agent. The proposed formulation also combines folic acid (FA) and iRGD peptides as targeting ligands to enhance tumor cell specificity and penetration, while the pH-sensitive liposomes ensure the controlled drug release in the acidic tumor microenvironment. The physicochemical characterization revealed that 5-FU-iRGD-FA-pSL possesses optimal size, low polydispersity index, and favorable zeta potential, enhancing its stability and targeting capabilities. In vitro studies demonstrated significantly enhanced cellular uptake, cytotoxicity, and inhibition of cell migration in MCF-7 BC cells compared to free 5-FU and non-targeted liposomal formulations. DAPI staining revealed significant apoptotic features, including chromatin condensation (CC) and nuclear fragmentation (NF), with 5-FU-iRGD-FA-pSL inducing more pronounced apoptosis compared to 5-FU-pSL. Furthermore, in vivo analysis in a BC rat model showed superior anti-tumor efficacy, reduced systemic toxicity, and improved safety profile of the 5-FU-iRGD-FA-pSL formulation. This dual-targeting pSL system presents a promising approach for enhancing the therapeutic index of 5-FU, offering a potential strategy for more effective BC treatment.

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1. Introduction

Breast cancer (BC) poses a serious threat to women's health and is known as the most common cancer worldwide.^1,2 According to the American Cancer Society (ACS), there will be 310 720 new cases of invasive BC and 55 500 new cases of ductal carcinoma in situ (DCIS) in the United States in 2024, resulting in approximately 42 250 deaths.³ BC is a heterogeneous disease with complex molecular profiles, presenting significant treatment challenges. In current clinical practice, chemotherapy remains one of the primary treatments for BC, particularly for aggressive or metastatic forms of the disease. One such chemotherapy agent widely used in the treatment of BC is 5-fluorouracil (5-FU), a pyrimidine analogue that interferes with DNA synthesis and induces apoptosis in rapidly dividing cancer cells.⁴ Despite its effectiveness, the non-selective nature of 5-FU means that it also targets healthy proliferating cells, which can lead to severe side effects such as gastrointestinal toxicity, myelosuppression, and hand-foot syndrome. These systemic toxicities limit the therapeutic dose that can be administered and hinder the overall efficacy of the treatment.^5–8 This has spurred significant interest in developing more targeted delivery systems to minimize these side effects while maximizing the therapeutic impact on cancer cells.

To overcome the limitations, a promising strategy for improving the solubility and delivery of chemotherapy drugs is using liposomes, which are spherical vesicles made from lipid bilayers.⁹ Liposomes are well-established in drug delivery due to their ability to encapsulate various hydrophilic and hydrophobic drugs, providing enhanced solubility, stability, and controlled release. In addition, liposomes have the advantage of biocompatibility, which helps reduce the immunogenicity and toxicity commonly associated with other drug delivery systems. The properties of liposomes, such as size, surface charge, and rigidity, are influenced by their lipid composition.^10–13 For example, saturated phospholipids like diphosphatidylcholine (DPPC) tend to produce more rigid bilayers, increasing liposome stability and reducing encapsulated drug leakage.¹⁴ On the other hand, unsaturated phospholipids can create softer and more flexible membranes, which may facilitate drug release under specific conditions, such as in acidic tumor environments.

Liposomes can also be modified to improve their pharmacokinetics. For example, polyethylene glycol (PEG)-modified liposomes, such as DSPE-PEG2000, have been shown to significantly extend the circulation time of liposomes in the bloodstream by reducing their clearance via the reticuloendothelial system (RES).^15,16 Recent studies have demonstrated that PEGylation can lead to the production of anti-PEG IgM and IgG antibodies, which may activate complement pathways and accelerate the clearance of PEGylated nanoparticles upon subsequent doses. These immune responses may also contribute to hypersensitivity reactions in some patients.^17–19 To overcome these limitations, various strategies have been explored, including the use of alternative stealth polymers, such as zwitterionic polymers (e.g., poly(carboxybetaine) or poly(sulfobetaine)), which exhibit reduced immunogenicity compared to PEG.^20,21 Additionally, optimizing PEG's density and molecular weight on the liposomal surface can minimize the likelihood of immune activation.²² Another promising approach is the use of biodegradable or cleavable PEG linkers that detach in response to specific stimuli (e.g., pH, enzymes, or redox conditions) within the tumor microenvironment, reducing prolonged immune exposure to PEG. One of the most famous and clinically successful examples of PEGylated liposomal formulations is Doxil®, a liposomal formulation of the chemotherapeutic agent doxorubicin. Doxil® is frequently prescribed for treating various cancers, including BC. It has demonstrated the ability to lower the risk of drug-induced cardiotoxicity and decrease gastrointestinal side effects like nausea and vomiting, especially when compared to standard free doxorubicin. The liposomal formulation of doxorubicin effectively limits its delivery to the heart and gastrointestinal tissues, thus mitigating these common side effects. However, despite the advantages, liposomal drug delivery still faces significant challenges. These include issues with delayed drug release, inefficient drug delivery to target tissues, and insufficient cellular uptake after internalization into the endosomal compartment.^11,23

To address these limitations, the development of 5-FU-pSLs offers a promising strategy, especially in cancer therapy. Tumor microenvironments (TMEs) are typically more acidic than healthy tissues due to the increased rate of anaerobic glycolysis and CO₂ accumulation, which reduces extracellular pH.²⁴ While normal tissues maintain a pH of around 7.4, tumors often exhibit pH levels between 5.5 to 7.0. This pH difference creates an opportunity to design liposomes that remain stable in the neutral pH of healthy tissues but destabilize and release their drug payload upon encountering the tumour's acidic environment. This approach minimizes drug leakage in the bloodstream and ensures that the therapeutic agents are explicitly released at the tumor site.²⁵

A key component of 5-FU-pSLs is dioleoylphosphatidylethanolamine (DOPE), a lipid that undergoes a phase transition when exposed to an acidic environment. In the neutral pH of the bloodstream, DOPE is stable within the lipid bilayer. However, in acidic conditions, the reduced hydration of DOPE's polar headgroup leads to non-lamellar structure formation, such as inverted hexagonal phases, that disrupt the bilayer and facilitate drug release.²⁶ This phase transition improves drug delivery by enabling the release of the encapsulated drug into the cytoplasm once the liposome is internalized by the cell and undergoes endosomal acidification. To stabilize the liposomes under neutral pH conditions, stabilizing lipids like cholesteryl hemisuccinate (CHEMS) or phosphatidylserine can be incorporated. In an acidic environment, however, these stabilizing lipids undergo protonation, which weakens their ability to stabilize the bilayer, thus triggering the release of the drug payload.²⁷

Beyond pH sensitivity, liposomes can also be modified with specific targeting ligands to enhance their uptake by cancer cells and improve treatment specificity. Folate receptors (FR), often overexpressed on the surface of cancer cells, including BC, are a common target for liposome functionalization. Folate-conjugated liposomes exhibit selective binding to FR where FA is incorporated on their surface. This targeted delivery system promotes enhanced internalization by tumor cells while minimizing interaction with healthy, non-target cells.²⁸ For instance, liposomes conjugated with folate (FA) and loaded with doxorubicin have demonstrated a marked improvement in drug delivery to breast tumor cells, leading to better therapeutic outcomes than non-targeted or free drug formulations.²⁹

The iRGD peptide represents another promising ligand for improving liposome targeting. It specifically binds to αvβ3 integrins, which are commonly expressed on the surface of endothelial and tumor cells.³⁰ Upon binding to αvβ3, iRGD also interacts with neuropilin-1 (NRP-1), triggering the tumor-penetrating peptide (TPP) effect. This interaction enhances the ability of liposomes to penetrate the tumor microenvironment, facilitating deeper tissue infiltration. Incorporating iRGD to promote tumor penetration has demonstrated favorable outcomes in various cancer models, and its combination with FA-based targeting strategies could further enhance the specificity and therapeutic efficacy of liposomal drug delivery systems.³⁰

We developed the 5-FU-iRGD-FA-pSL formulation in a previous investigation, utilizing DSPE–PEG2000–FA and DSPE–PEG2000–iRGD conjugates. This formulation exhibited a significantly enhanced release of 5-FU at an acidic pH of 5.5 compared to the physiological pH of 7.4, supporting its potential for selective targeting of tumor sites. Moreover, the 5-FU-iRGD-FA-pSL demonstrated increased cytotoxicity against SK-BR-3 and MDA-MB-231 BC cell lines relative to both the 5-FU-pSL and free 5-FU treatments. The formulation demonstrated exceptional long-term stability, preserving its structural integrity under in vitro conditions replicating biological fluids’ characteristics.³¹

These promising results laid the groundwork for further exploring the anti-tumor activity of this novel liposomal formulation in human cancer models. This study aims to evaluate the efficacy of 5-FU-iRGD-FA-pSL in MCF-7 metastatic BC cells through in vitro assays assessing cell uptake and cytotoxicity. Additionally, the safety profile of this liposomal formulation was assessed through acute toxicity testing in healthy Wistar rats, utilizing histological and laboratory analyses to ensure its potential for future clinical translation.

2. Experimental section

2.1 Materials and methods

5-Fluorouracil (5-FU) was procured from HiMedia (Mumbai, India). Phospholipids (80% phosphatidylcholine, E80) were provided by Lipoid GmbH (Ludwigshafen, Germany), while CHEMS and dioleoylphosphatidylethanolamine (DOPE) were sourced from ChemScence (Burlington, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI), Fluorescein Isothiocyanate (FITC), and Rhodamine B dyes were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’ Modified Eagle's Medium (DMEM), PSA antibiotics, and trypsin were procured from Invitrogen (Thermo Fisher Scientific, São Paulo, Brazil), and Fetal bovine serum (FBS) was from Gibco (São Paulo, Brazil). DSPE-PEG2000-FA and DSPE-PEG2000-iRGD were synthesized in our laboratory. All other chemicals were of analytical grade. The MCF-7 cell line was obtained from the National Centre for Cell Science (NCCS), Pune, India.

2.2 Preparation and physicochemical characterization of 5-FU-pSLs

The 5-FU-pSLs were prepared using the thin-film hydration method, as previously optimized In our lab.³¹ Lipid mixtures consisting of E-80, DOPE, CHEMS, and DSPE-PEG2000 were combined in precise molar ratios. The lipids and drug were dissolved in a 9 : 1 chloroform:methanol solvent mixture, and the organic solvent was then removed by rotary evaporation under reduced pressure, yielding a thin lipid film. To promote complete ionization of CHEMS, NaOH was added at a 1 : 1 ratio with CHEMS, and the mixture was vigorously shaken to achieve a multilayered structure. Liposomes were then sonicated in an ice bath at 40% amplitude for 2 minutes using a probe sonicator (PRO900, Labman Scientific Instruments, Chennai, India). The unencapsulated drug was separated by centrifugation at 4000 rpm for 10 minutes at 4 °C, and the resulting 5-FU-pSLs were stored at 4 °C. For dual-ligand surface-modified, 5-FU-pSLs, DSPE–PEG₂₀₀₀–FA and DSPE–PEG₂₀₀₀-iRGD conjugates were added in a 1 : 1 molar ratio, following the same preparation procedure.

The size and polydispersity index (PDI) of the 5-FU-pSLs were measured by dynamic light scattering (DLS) at 25 °C using a Zetasizer NanoZS90 (Malvern Instruments, Worcestershire, UK), with measurements were performed at a 90° scattering angle. Samples were diluted in 0.9% NaCl solution (1 : 100) and analyzed in triplicate. Transmission electron microscopy (TEM) was employed to assess the surface morphology of the liposomes, following established protocols.

2.3 Entrapment efficiency (% EE) and drug loading (%DL)

The % EE and % DL of the developed 5-FU-pHLip and FA-iRGD-5-FU-pHLip were assessed using the high-performance liquid chromatography (HPLC) method.³² To do this, a predetermined volume of liposomal solution (500 μL) was centrifuged at 2000 rpm for 5 min using Nanosep® (Pall Corp., Exton, PA, US) advanced centrifugal tubes in order to separate the unentrapped drug. A 1% solution of Triton X-100 was used to disrupt the bilayer of the liposomes and subsequently passed through a 0.22 μm filter. Afterward, the solution was diluted by adding a mobile phase consisting of a mixture of water and methanol in a ratio of 98 : 02 (v/v). The diluted solution was then injected into a C18 column (250 mm × 4.6 mm i.d., with 5 μm) (Shimadzu, Corp., Kyoto, Japan). The elution process lasted 5 min, with a 1 mL min⁻¹ flow rate. Detection was conducted using a UV detector (Shimadzu Prominence-i LC-2030 Plus, Shimadzu, Corp., Kyoto, Japan) set at a wavelength of 266 nm. The values for % EE and %DL were determined using the following equation.

%EE = (Weight of entrapped drug)/(Initial weight of drug added) ×100

%DL = (Weight of entrapped drug)/(Weight of entrapped drug + Weight of lipids used in the liposomes preparation) × 100

2.4 In vitro drug release

The pH sensitivity of the liposomal formulations (5-FU-pSLand 5-FU-iRGD-FA-pSL) was assessed employing the dialysis bag method. Briefly, dialysis bags with a molecular weight cutoff of 12 kDa were filled with 5 mL of liposomal suspension. These bags were then incubated in two different release media: Phosphate-buffered saline PBS (pH 7.4)–methanol (7 : 3) and PBS (pH 5.0)–methanol (7 : 3). Both release media contained 0.1% (v/v) Tween 80 to ensure that there was a sufficient amount of liposomal suspension available for diffusion. The dialysis bags were placed in flasks and agitated on an orbital shaker at 200 rpm, at 37 ± 2 °C. At specified time intervals (0, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 72 h), 500 μL samples were taken from the release media and replaced with an equivalent fresh medium. The quantity of 5-FU released was quantified using the previously reported HPLC method.³³

2.5 In vitro biological stability assessment

To evaluate the biological stability of the developed FA-iRGD-5-FU-pHLips liposomal suspension, it was separately incubated in two different media: PBS (pH 7.4) and DMEM, both supplemented with 5% (v/v) FBS. This step was intended to simulate the conditions in both in vitro and in vivo biological environments. The liposomal suspension was diluted at a 1 : 4 ratio in PBS or DMEM. The mixture was then incubated at 37 °C for 24 hours. Samples were taken both prior to and following the 24-hour incubation period. The particle size and zeta potential were measured using previously established methodologies to assess any changes in the liposomes’ physical properties.

2.6 Cell culture

The MCF-7 BC cell line was sourced from the NCCS, in Pune, India. Cells were cultured in DMEM (Himedia, Mumbai, India) supplemented with 10% heat-inactivated fetal bovine serum, 100 U mL⁻¹ penicillin–streptomycin, and a 100 mg mL⁻¹ ampicillin antibiotic mixture (all from Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The cells were incubated in a humidified environment at 37 °C with 5% CO₂.

Cytotoxicity of blank 5-FU-pSL formulations, free 5-FU, 5-FU-pSL, and 5-FU-iRGD-FA-pSL was assessed using the MTT assay on MCF-7 BC cells over a 48-hour incubation period. A cell suspension (2 × 10⁴ cells per well) was seeded in 96-well plates and cultured in DMEM supplemented with 10% FBS and 1% antibiotic solution (penicillin–streptomycin) for 24 hours at 37 °C in a 5% CO₂ atmosphere. Following the incubation, the cells were treated with various concentrations (0.1–50 μg mL⁻¹) of the formulations, prepared in culture medium. The cells were then incubated for an additional 24 hours. After treatment, 20 μL of MTT solution (5 mg mL⁻¹) was added to each well and incubated for 2 hours at 37 °C. Following incubation, the culture medium was carefully removed, and the formazan crystals formed in the cells were dissolved in 100 μL of dimethyl sulfoxide (DMSO). The plates were agitated for 20 minutes to ensure complete dissolution of the crystals. The absorbance was measured at 540 nm using a microplate reader (iMark, Bio-Rad, Hercules, CA). All experiments were performed in triplicate. The cell viability was determined based on the optical density values, with untreated cells used as the control.^34–37

2.7 Cell migration

MCF-7 BC cells were plated in 12-well plates at a density of 3 × 10⁵ cells per well and incubated at 37 °C for 24 hours to establish a confluent monolayer. A wound was then introduced in the cell monolayer using a sterile 10 μL pipette tip (n = 3), and initial images were captured to record the wound area. After wounding, the cells were treated with 1 mL of fresh DMEM supplemented with 1% (v/v) FBS, containing either free drug or 5-FU-pSL formulations. The plates were incubated at 37 °C for an additional 48 hours to allow for wound closure. To ensure consistency, images of the same wound were taken before and after treatment for each well.³⁸ The degree of wound closure was evaluated using the MRI Wound Healing Tool plugin in ImageJ 1.45 (National Institutes of Health, Bethesda, MD, USA). The wound closure rate was calculated by measuring the reduction in wound area over time.^39,40

2.8 DAPI staining

To evaluate nuclear changes associated with apoptosis, DAPI staining was performed. Briefly, MCF-7 cells were rinsed thrice with ice-cold PBS and then incubated at room temperature for 5 minutes. The cells were then treated with a DAPI solution (2 mg mL⁻¹ in PBS) and incubated for 15 minutes to allow for nuclear staining. After staining, nuclear alterations indicative of apoptosis were observed using a fluorescence microscope (Ti-S Nikon Eclipse), and images were captured for further analysis.^41,42

2.9 Liposomal cellular uptake by Confocal Microscopy

For cellular uptake analysis, MCF-7 cells were plated in 12-well plates with glass coverslips at an initial density of 2 × 10⁵ cells per well and incubated overnight. Coumarin-6-loaded and Rhodamine-B-loaded placebo pH-sensitive liposomes were then added to the cells and incubated for 2 hours at 37 ± 0.5 °C in a 5% CO₂ atmosphere. After incubation, the formulations were removed, and cells were washed thrice with PBS (pH 7.4). The cells were fixed with 4% paraformaldehyde for 30 minutes, then permeabilized with 0.1% Triton X-100 for 5 minutes to facilitate DAPI staining. Nuclei were stained with DAPI (5 μg mL⁻¹) for 10 minutes, followed by three PBS washes. The coverslips were mounted onto glass slides, and cellular internalization of the formulations was observed using a confocal laser scanning microscope (LSM900, Zeiss, Germany) at 20× magnification.^43,44

2.10 In vivo studies

An in vivo study was conducted using female Albino Wistar rats, following the ethical guidelines approved by the Institutional Animal Ethics Committee (IAEC) at BBDNIIT (Approval No. BBDNIIT/IAEC/2020/11). The animals were randomly assigned to five experimental groups (n = 6), each receiving a single dose of pure 5-FU, 5-FU-pSL, or 5-FU-iRGD-FA-pSL at 10 mg kg⁻¹. The control group was treated with saline (NaCl, 0.9% w/v). The tumor was induced by administering a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) (8 mg kg⁻¹) via tail vein injection. with treatment administered for 14 days following carcinogen exposure. Throughout the study, animals were regularly monitored for changes in body weight and survival. The variation in body weight was determined by subtracting the initial weight from the final weight. Tumor progression was assessed through periodic body weight monitoring and histopathological analysis. Mammary gland tissues were examined using hematoxylin and eosin (H&E) staining to observe morphological changes.⁴⁵ The biosafety of the formulations was evaluated in various organs, including the kidneys, liver, spleen, and lungs, also using H&E staining.⁴⁶ Additionally, blood samples were collected from the animals, and enzyme-linked immunosorbent assays (ELISA) were used to measure the levels of inflammatory cytokines, including IL-6, IL-1β, MMP-1, and TNF-α.

2.11 Measurement of body weight variation

The body weight of all animals was systematically monitored at designated time intervals, and a graph was constructed to depict weight fluctuations over time for each group throughout the study period.⁴⁷

2.12 Blood chemistry analysis

Hematological parameters, including levels of hemoglobin (HGB), white blood cells (WBC), red blood cells (RBC), and platelet count, were evaluated for each group. For biochemical analysis, blood samples were centrifuged at 1100 × g for 15 minutes, and the plasma was stored at −20 °C for subsequent analysis. Kidney function was assessed by measuring serum creatinine and urea levels, while liver function was determined by evaluating the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).^48,49 All analyses were performed using a Bioplus BIO-2000 semiautomatic analyzer with commercial assay kits.

2.13 Histopathological analysis

Tissues from the mammary glands, liver, kidneys, spleen, and heart were collected for histopathological examination. The samples were fixed in 10% buffered formaldehyde (v/v) for 24 hours, followed by dehydration through a graded alcohol series and embedding in paraffin. Thin sections, 4 μm in thickness, were prepared using a microtome and stained with hematoxylin and eosin (H&E) according to standard protocols.^42,50,51 For detailed analysis and imaging, the stained sections were examined under an optical microscope (Olympus BX-40; Olympus, Tokyo, Japan).

2.14 Pro-inflammatory cytokines assessment

Breast tissue samples were evaluated for elevated levels of pro-inflammatory cytokines utilizing commercially available ELISA kits, in accordance with the manufacturer's guidelines.^52,53

3. Results and discussion

3.1 Physicochemical characterization

The physicochemical properties of the liposomal formulations (5-FU-pSL and 5-FU-iRGD-FA-pSL) are shown in Table 1. The DLS analysis revealed no significant differences in size, PDI, or zeta potential between the 5-FU-pSL and 5-FU-iRGD-FA-pSL formulations (Fig. 1). The particle sizes of both formulations were similar, with 5-FU-pSL and 5-FU-iRGD-FA-pSL measuring approximately 133.83 ± 4.15 nm and 138.66 ± 3.27 nm, respectively. Both formulations exhibited low PDI values (0.248 for 5-FU-pSL and 0.10 for 5-FU-iRGD-FA-pSL), which indicates a narrow size distribution and a monodispersed nature. Additionally, both formulations showed near-neutral zeta potentials of −12.2 mV and −14.7 mV, indicating a neutral surface charge. The pH of all formulations remained close to neutral after preparation. Transmission electron microscopy (TEM) analysis further supported these findings, showing that 5-FU-iRGD-FA-pSL predominantly consisted of unilamellar, spherical structures with sizes under 200 nm, confirming the nanoscale size and structural integrity of the liposomes.

Table 1 Physicochemical characterization of 5-FU-pSL and 5-FU-iRGD-FA-pSL formulations

Formulation	Particle size (nm)	PDI	Zeta potential (mV)	EE (%)	Drug loading (%)
5-FU-pSL	133.83 ± 4.15	0.248	−12.2 ± 0.35	86.57 ± 2.41	6.19 ± 0.97
5-FU-iRGD-FA-pSL	138.66 ± 3.27	0.103	−14.7 ± 0.21	92.75 ± 3.01	7.42 ± 1.26

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Fig. 1 Particle size intensity distribution of 5-FU-pSL and 5-FU-iRGD-FA-pSL (A), zeta potential (B) and TEM (C) of 5-FU-iRGD-FA-pSL, respectively.

% EE was notably higher for the 5-FU-iRGD-FA-pSL formulation (92.75 ± 3.01%) compared to the 5-FU-pSL formulation (86.57 ± 2.41%). The improved EE in the decorated liposomes can be attributed to the additional interactions provided by the ligands, which may enhance drug encapsulation stability. Similarly, %DL was enhanced in the dual-decorated formulation (7.42 ± 1.26%) compared to the non-decorated liposomes (6.19 ± 0.97%), highlighting the ability of the functionalized carrier to accommodate a higher drug payload.

3.2 In vitro drug release study

The in vitro drug release profiles of 5-FU from free 5-FU, 5-FU-pSL, and FA-iRGD-5-FU-pSL formulations under physiological (pH 7.4) and acidic (pH 5.5) conditions are shown in Fig. 2. Free 5-FU exhibited a rapid and nearly complete release at both pH 7.4 and pH 5.5 within 8 h. In contrast, the 5-FU-pSL showed a slower and sustained release profile. At pH 7.4, the cumulative release was significantly lower after 72 h, indicating excellent stability under physiological conditions. However, at pH 5.5, a marked increase in release was observed, reaching approximately 80% at 72 h. This enhanced release at acidic pH is due to the pH-responsive nature of the liposomal membrane, where components such as CHEMS and DOPE destabilize the bilayer structure in the acidic tumor microenvironment, facilitating efficient drug release.

Fig. 2 In vitro drug release profile of free 5-FU drug, 5-FU-pSL and FA-iRGD-5-FU-pHLip at pH 7.4 and 5.5.

The FA-iRGD-5-FU-pSL displayed a similar pH-sensitive release pattern but slightly higher drug release than 5-FU-pSL. At pH 7.4, the cumulative release was below 30% after 72 h, confirming its stability in circulation. At pH 5.5, the release was more pronounced, reaching nearly 85% at 72 h. This can be attributed to the enhanced pH sensitivity of the formulation, coupled with the targeting capabilities of the FA and iRGD ligands, which may facilitate membrane destabilization and promote efficient drug diffusion under acidic conditions.

The release kinetics of pSL formulations (5-FU-pSL and 5-FU-iRGD-FA-pSL) were evaluated at pH (7.4 and 5.5) conditions to mimic systemic circulation and tumor microenvironments, respectively (Table 2). Both formulations exhibited controlled release profiles at pH 7.4, with release kinetics fitting the Korsmeyer–Peppas and Higuchi models, indicating a diffusion-dominated mechanism. An enhanced release was observed at acidic pH, particularly for 5-FU-iRGD-FA-pSL, which showed the highest R² values for the Korsmeyer–Peppas and Higuchi models, highlighting its superior pH responsiveness. The release at pH 5.5 is attributed to the protonation of CHEMS, causing destabilization of the lipid bilayer, while the structural integrity at pH 7.4 was maintained by DSPE-PEG2000, minimizing premature release. The dual surface decoration with iRGD and FA ligands ensured targeted delivery and controlled release in both conditions, with superior performance compared to non-decorated liposomes. These findings demonstrate that 5-FU-iRGD-FA-pSL effectively combines stability in systemic circulation with enhanced tumor-specific release, validating its potential as an advanced drug delivery system for cancer therapy.

Table 2 Correlation coefficients (R²) for various kinetic models to fix the best fit by using DD solver software

Formulation	Zero-order	First-order	Higuchi	Korsmeyer–Peppas	Hixson–Crowell
5-FU-pSL (pH 7.4)	0.7755	0.8636	0.9514	0.9841	0.8068
5-FU-pSL (pH 5.5)	0.7870	0.9660	0.9589	0.9864	0.4678
5-FU-iRGD-FA-pSL (pH 7.4)	0.8104	0.8724	0.9698	0.9824	0.9211
5-FU-iRGD-FA-pSL (pH 5.5)	0.8073	0.9422	0.9641	0.9825	0.5714

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3.3 In vitro biological stability

The biological stability of 5-FU-pSL and 5-FU-iRGD-FA-pSL was assessed in PBS (pH 7.4) and DMEM media over 24 h (Fig. 3). The mean particle size of both formulations remained stable with minor fluctuations, indicating their structural integrity in both physiological and cell culture conditions. The slight increase in particle size observed over time could be attributed to potential aggregation or biomolecular adsorption. Notably, the 5-FU-iRGD-FA-pSL formulation displayed a slightly larger particle size compared to 5-FU-pSL, consistent with the added surface modifications.

Fig. 3 In vitro biological stability of (5-FU-pSL) and 5-FU-iRGD-FA-pSL assessed in (A) PBS (pH 7.4); (B) DMEM media over 24 h.

The zeta potential of both formulations exhibited minimal variations over time, with a slight reduction. This trend may result from the interaction of the liposomal surface with ions and proteins in the media, leading to partial neutralization of surface charge. The more negative zeta potential of 5-FU-iRGD-FA-pSL suggests improved colloidal stability due to enhanced electrostatic repulsion. Interestingly, the stability of the formulations in DMEM was superior to that in PBS. The presence of proteins and nutrients in DMEM likely contributed to forming a stabilizing protein corona around the liposomes, reducing aggregation and maintaining consistent particle size and zeta potential. In PBS, the absence of such proteins may have resulted in slightly greater variability in particle size. These results suggested the excellent stability of both 5-FU-pSL and 5-FU-iRGD-FA-pSL in physiological and cell culture environments. Incorporating iRGD and FA ligands facilitates targeted delivery and enhances the stability of the formulations by mitigating rapid degradation or aggregation.

3.4 In vitro cytotoxicity

The in vitro cytotoxicity of the developed liposomal formulations was evaluated using MCF-7 BC cells through an MTT assay to assess cell viability at varying concentrations (Fig. 4(A) and (B)). As shown in Fig. 2(A), blank 5-FU-pSL and 5-FU-iRGD-FA-pSL exhibited minimal cytotoxic effects on MCF-7 cells across all tested concentrations (0–20 μM). Cell viability remained above 95% in both formulations, indicating that these carriers are inherently biocompatible and do not contribute to cytotoxicity. This high level of cell viability suggests that blank 5-FU-pSL formulations does not showed any toxic effects on non-targeted cells.

Fig. 4 Cytotoxicity of the treatments blank 5-FU-pSL (A), free 5-FU, 5-FU-iRGD-FA-pSL (B) against MCF-7 cell lines. Microscopic images of wound healing assay (C) and % cell migration plot (D) on MCF-7 cell lines. Data are expressed by the mean ± SD of the mean of independent experiments (n = 3). Migration studies and plot (C) and (D). [One asterisk (*), two asterisks (**), and three asterisks (***) denote significant p values, i.e., <0.05, <0.01, and <0.001, respectively, and ns as non-significant].

The cytotoxicity of free 5-FU, 5-FU-pSL, and 5-FU-iRGD-FA-pSL was tested at various concentrations (0–20 μg mL⁻¹) to evaluate their efficacy against MCF-7 cells for 48 h. At lower concentrations (1 and 5 μg mL⁻¹), cell viability was only slightly reduced across all formulations, with 5-FU-iRGD-FA-pSL showing the greatest decrease in viability, though not statistically significant at 1 μg mL⁻¹. At higher concentrations (10, 15, and 20 μg mL⁻¹), a marked decrease in cell viability was observed, with 5-FU-iRGD-FA-pSL demonstrating the most significant cytotoxicity compared to both free 5-FU and 5-FU-pSL (p < 0.01 at 10 μg mL⁻¹, p < 0.001 at 15 and 20 μg mL⁻¹). This indicates that the FA-iRGD decoration enhances the efficacy of the 5-FU-loaded liposomes, likely due to improved cellular uptake mediated by FA and integrin receptor targeting. The results demonstrate that 5-FU-iRGD-FA-pSL effectively enhances the anticancer effects of 5-FU against MCF-7 cells, achieving higher cytotoxicity than free 5-FU alone and non-decorated liposomes at higher concentrations.

3.5 Cell migration

Cell migration was assessed using the wound healing assay in MCF-7 cells, and the results are presented in Fig. 4(C) and (D). The 5-FU-iRGD-FA-pSL formulation significantly inhibited cell motility. After 48 hours of treatment, only 17.61 ± 2.47% of the wound area was closed in cells treated with 5-FU-iRGD-FA-pSL, compared to higher wound closure in cells treated with free 5-FU and 5-FU-pSL. These findings suggest that FA-iRGD surface modification enhances the ability of pH-sensitive liposomes to inhibit cell migration. The enhanced effect is likely due to the dual-targeting action of FA and iRGD, which promotes increased cellular uptake and disrupts key signaling pathways involved in migration. Therefore, the 5-FU-iRGD-FA-pSL formulation is more effective in reducing tumor cell motility, suggesting potential anti-metastatic activity.

3.6 DAPI staining

DAPI staining, a common nuclear counterstain, revealed significant changes in nuclear morphology following treatment with 5-FU-pSL and 5-FU-iRGD-FA-pSL formulations in MCF-7 cells (Fig. 5). Untreated cells exhibited normal, intact nuclear structures. However, apoptotic features such as NF and CC were observed after treatment with both liposomal formulations. Nuclear morphology analysis identified viable cells (orange arrow), CC (red arrow), NF (yellow arrow), and apoptotic bodies. Notably, the 5-FU-iRGD-FA-pSL formulation induced more pronounced apoptotic changes, with a higher incidence of fragmented nuclei and condensed chromatin compared to 5-FU-pSL.

Fig. 5 DAPI staining revealed significant changes in the morphology of MCF-7 cell nuclei following treatment with 5-FU-pSL and 5-FU-iRGD-FA-pSL formulations. Apoptotic features, including viable cells (VC), chromatin condensation (CC), and nuclear fragmentation (NF), were observed, as indicated by arrows.

3.7 Cellular uptake

Fluorescence microscopy analysis revealed that both 5-FU-pSL and 5-FU-iRGD-FA-pSL formulations were efficiently internalized by MCF-7 cells. However, the uptake of 5-FU-iRGD-FA-pSL was significantly higher compared to 5-FU-pSL, as evidenced by increased fluorescence intensity in cells treated with the FA-iRGD-modified liposomes (Fig. 6). Notably, the fluorescence was concentrated in the perinuclear region, suggesting enhanced cellular uptake and subsequent intracellular trafficking. This increased uptake is likely attributed to the dual-targeting effect of the FA and iRGD ligands, which specifically bind to FR and αvβ3 integrins, respectively (Fig. 6(A)). These receptors are overexpressed on the surface of MCF-7 cells, facilitating more efficient internalization of the liposomal formulation.

Fig. 6 Cellular internalization of Coumarin-6 (A) and Rhodamine-B (B) loaded 5-FU-pSL formulation on MCF-7 cell lines with mean florescence intensity.

Similar trends were observed with Rhodamine-B-labelled formulations. Cells treated with 5-FU-iRGD-FA-pSL exhibited stronger fluorescence signals within the cytoplasm, indicating a higher level of Rhodamine-B accumulation compared to cells treated with 5-FU-pSL (Fig. 6(B)). This suggests that the dual-targeting strategy not only enhances the uptake of the encapsulated drug but also improves the overall intracellular accumulation of liposomal contents.

3.8 In vivo acute toxicity

3.8.1 Body weight and tumor burden. In the groups treated with the toxicant, the induction and progression of mammary gland cancer were evaluated through changes in animal weight, the total tumor burden, and histopathological analysis. The body weight variation observed among experimental groups highlights the systemic toxicity, tumor progression, and therapeutic efficacy of the tested formulations (Fig. 7(A)). Initial body weights across all groups were consistent, ranging from 165 ± 5 g to 195 ± 5 g, with the control group maintaining stable weight throughout the study (195 ± 5 g). This stability in the control group underscores the absence of adverse physiological effects, establishing a baseline for evaluating treatment impacts. Significant weight loss was observed in the toxic control group (40 ± 5 g, 20.5% decrease), indicative of pronounced systemic toxicity due to the toxicant's detrimental effects on metabolic health and tissue integrity. Similarly, animals treated with free 5-FU experienced moderate weight loss (15 ± 3 g), attributed to the known systemic toxicity of 5-FU, such as gastrointestinal side effects and cachexia, stemming from non-specific drug distribution and off-target effects. Conversely, animals treated with 5-FU-pSL exhibited reduced weight loss (8 ± 2 g), reflecting improved tolerability due to the selective release of 5-FU in the acidic tumor microenvironment, which minimizes systemic exposure. The 5-FU-iRGD-FA-pSL further reduced weight loss to a minimal level (5 ± 2 g), closely resembling the control group. The superior performance of these dual-decorated liposomes is attributed to the tumor-specific targeting facilitated by iRGD and FA ligands, which enhance receptor-mediated delivery and reduce off-target toxicity.

Fig. 7 Animal weight variation (A), tumour burden (anti-tumor activity) (B) after treatment with 5-FU-pSL formulations.

The stability of the control group's weight indicates that tumor burden alone did not significantly influence weight loss. The substantial weight reduction in the toxic control and free 5-FU groups suggests that drug-induced toxicity was the dominant factor. The progressive mitigation of weight loss from free 5-FU to 5-FU-pSL and 5-FU-iRGD-FA-pSL groups underscores the advanced formulations’ ability to reduce systemic side effects effectively.

Tumor burden analysis revealed significant differences in therapeutic efficacy (Fig. 7(B)). The toxic control group exhibited a 175 ± 15% increase in tumor burden, while conventional 5-FU treatment resulted in a 125 ± 8% increase. The 5-FU-pSL formulation reduced tumor burden to 65 ± 5%, and the 5-FU-iRGD-FA-pSL formulation demonstrated the most significant effect, with only a 15 ± 3% tumor burden (p < 0.001 compared to all other groups). Statistical analysis showed that 5-FU-iRGD-FA-pSL achieved an 88.6% reduction in tumor burden compared to the toxic control (p < 0.001), surpassing the reductions observed with pure 5-FU (28.6%) and 5-FU-pSL (62.9%). These results suggest that both 5-FU-pSL and 5-FU-iRGD-FA-pSL formulations effectively reduced tumor burden, with the latter showing the most potent anti-tumor activity, highlighting the potential of targeted liposomal delivery systems to enhance chemotherapy efficacy.

3.8.2 Biochemical analysis of hepatorenal toxicity. The hepatorenal toxicity of different 5-FU formulations was evaluated by analyzing renal (creatinine and urea) and hepatic (ALT and AST) function markers across treatment groups (Table 3). The 5-FU group exhibited significant elevations in serum creatinine and blood urea levels, indicating renal impairment. In contrast, 5-FU-pSL and 5-FU-iRGD-FA-pSL formulations, showed creatinine and urea levels similar to the control group, demonstrating enhanced renal safety. Notably, 5-FU-iRGD-FA-pSL exhibited the lowest creatinine levels among the treatment groups, suggesting its superior ability to mitigate renal toxicity. The improved renal safety observed with these formulations may be attributed to the targeted drug delivery, which potentially reduces systemic distribution and nephrotoxic effects.

Table 3 Biochemical parameters of DMBA-induced rats after different treatments at dose of 10 mg kg⁻¹

	Control	5-FU	5-FU-pSL	5-FU-iRGD-FA-pSL
Data are expressed as mean ± SD (n = 6). All data were analyzed using a one-way ANOVA analysis of variance followed by Tukey's post-test. p < 0.05, **p < 0.01, ***p < 0.001 compared to control group ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 compared to 5-FU group ns: not significant compared to control group.
Creatinine (mg dL^−1)	0.40 ± 0.07	0.61 ± 0.08***	0.39 ± 0.05^ns	0.33 ± 0.07^ns,†
Urea (mg dL^−1)	51.47 ± 9.24	107.13 ± 17.46***	50.32 ± 6.27^ns,††	47.35 ± 7.44^ns,†
ALT (U L^−1)	30.62 ± 3.78	79.07 ± 13.65***	71.32 ± 2.94***	59.38 ± 88.45**,^†
AST (U L^−1)	90.4 ± 4.8	138.44 ± 12.6***	110.62 ± 2.4**^,†	103.1 ± 5.31*^,††

---

Liver enzyme levels (ALT and AST) were elevated in the 5-FU group, indicating hepatotoxicity. However, both targeted formulations, particularly 5-FU-iRGD-FA-pSL, showed significantly lower ALT and AST levels compared to conventional 5-FU treatment. The 5-FU-iRGD-FA-pSL group displayed the most favorable hepatic safety profile, with only moderate elevations in liver enzymes. This reduction in hepatotoxicity is likely due to the targeted nature of the formulation, which directs the drug more specifically to the tumor site, thus minimizing exposure to healthy liver tissue.

3.8.3 Hematological parameters. The hematological toxicity profiles of different 5-FU formulations were assessed by evaluating key blood parameters, including WBC count, RBC count, HGB levels, and platelet count (Table 4). The results revealed significant myelosuppression in the 5-FU group, characterized by a marked decrease in WBC and RBC counts and altered hemoglobin and platelet levels. In contrast, the targeted delivery systems, particularly 5-FU-iRGD-FA-pSL, showed notable improvements in preserving WBC and RBC counts, with values approaching normal levels. These formulations better maintained hematological parameters and demonstrated more balanced platelet counts compared to 5-FU treatment. Specifically, both liposomal formulations resulted in enhanced erythropoiesis and better platelet regulation, which could be attributed to their targeted drug delivery, reducing systemic toxicity. Overall, the targeted systems, especially 5-FU-iRGD-FA-pSL, exhibited a more favorable hematological safety profile, suggesting their potential to mitigate the hematotoxic effects of 5-FU while preserving essential blood cell functions.

Table 4 Hematological parameters of DMBA-induced rats after different treatments at dose of 10 mg kg⁻¹

	Control	5-FU	5-FU-pSL	5-FU-iRGD-FA-pSL
Data are expressed by the mean (n = 6) ± SD of the mean. All data were analyzed a one-way ANOVA analysis of variance followed by Tukey's post-test. p < 0.05, **p < 0.01, ***p < 0.001 compared to control group ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 compared to control group.
WBC (cell per mm^3 × 10^3)	6.4 ± 0.7	2.8 ± 0.3***	4.2 ± 0.9**^†	5.6 ± 1.2^ns,††
RBC (cell per mm^3 × 10^6)	4.4 ± 0.4	1.1 ± 0.3***	5.9 ± 0.4*^,†††	6.3 ± 0.4**^,†††
HGB (g dL^−1)	13.2 ± 1.1	14.8 ± 0.9*	12.9 ± 0.8^ns,†	11.6 ± 0.6*^,††
Platelets (cell per mm^3 × 10^3)	389 ± 91	597 ± 83***	523 ± 34**^,†	505 ± 31**^,†

---

3.8.4 Oxidative stress markers evaluations. To assess the protective effects of different drug formulations against DMBA-induced oxidative stress, key biomarkers were measured in treatment groups and compared to control and DMBA-treated toxic groups (Table 5).

Table 5 Serum levels and tissue content of MDA, and activities of SOD, catalase, and GSH in solid tumor tissue across the studied groups

	MDA (nmol mL^−1)	MDA (nmol per g^−1 tissue)	SOD (U mg^−1 per protein)	Catalase (U mg^−1 per protein)	GSH (μmol mg^−1 per protein)
Data are expressed as mean ± SD (n = 6). All data were analyzed by one-way ANOVA followed by Tukey's post-hoc test. **p < 0.01 compared to 5-FU and ***p < 0.001 compared to 5-FU and p < 0.01 compared to 5-FU-pSL.
5-FU	67.26 ± 6.47	134.27 ± 2.01	11.42 ± 1.95	12.07 ± 0.64	30.74 ± 1.74
5-FU-pSL	40.14 ± 5.49**	94.14 ± 4.745**	17.14 ± 1.53**	18.42 ± 0.42**	49.14 ± 0.42**
5-FU-iRGD-FA-pSL	31.79 ± 3.70***	84.60 ± 3.14***	24.84 ± 1.12***	26.41 ± 0.83***	80.09 ± 1.94***

---

Malondialdehyde (MDA), a marker of lipid peroxidation, was significantly elevated in the DMBA-treated toxic group compared to the control. In the 5-FU group, MDA levels were 67.26 ± 6.47 nmol mL⁻¹ in serum and 134.27 ± 2.01 nmol g⁻¹ in tissue, indicating considerable oxidative damage. The 5-FU-pSL formulation reduced MDA levels to 40.14 ± 5.49 nmol mL⁻¹ in serum and 94.14 ± 4.75 nmol g⁻¹ in tissue, suggesting some protective effect. Notably, the 5-FU-iRGD-FA-pSL group exhibited the lowest MDA levels of 31.79 ± 3.70 nmol mL⁻¹ in serum and 84.60 ± 3.14 nmol g⁻¹ in tissue, approaching values observed in the normal control group. Superoxide dismutase (SOD) activity was 11.42 ± 1.95 U mg⁻¹ protein in the 5-FU group, falling within the normal control range. Both the 5-FU-pSL and 5-FU-iRGD-FA-pSL groups showed significantly higher SOD activities, with values of 17.14 ± 1.53 U mg⁻¹ and 24.84 ± 1.12 U mg⁻¹, respectively, indicating enhanced antioxidant capacity in these groups.

Catalase activity was reduced in the 5-FU group (12.07 ± 0.64 U mg⁻¹), even lower than in the DMBA-toxic group. However, the 5-FU-pSL formulation showed improved catalase activity (18.42 ± 0.42 U mg⁻¹), and the 5-FU-iRGD-FA-pSL group exhibited the highest catalase activity (26.41 ± 0.83 U mg⁻¹) across all treatment groups. Compared to the control, reduced glutathione (GSH) levels were elevated in all treatment groups. The 5-FU group showed a GSH level of 30.74 ± 1.74 μmol mg⁻¹ protein, while the 5-FU-pSL and 5-FU-iRGD-FA-pSL groups exhibited 49.14 ± 0.42 μmol mg⁻¹ and 80.09 ± 1.94 μmol mg⁻¹, respectively, reflecting enhanced antioxidant defense. These results indicate the 5-FU-iRGD-FA-pSL formulation provided the most potent protection against DMBA-induced oxidative stress, as evidenced by the lowest lipid peroxidation, best preservation of antioxidant enzymes, and greatest enhancement of the body's natural antioxidant defenses.

3.8.5 Histopathological studies. Microscopic examination of breast tissue sections revealed distinct histological patterns across the experimental groups (Fig. 8). In the toxic group, histopathological assessment demonstrated invasive carcinoma of the left breast with significant infiltration into the surrounding adipose tissue and stroma, presenting characteristics reminiscent of fat necrosis. The cellular architecture exhibited predominantly isolated neoplastic cells dispersed irregularly throughout a densely fibrotic stromal matrix. The 5-FU treatment group showed persistent tumoral infiltration (red asterisks) into the adjacent adipose tissue. Notable features included marked nuclear pleomorphism and pronounced nuclear hyperchromasia, indicating sustained cellular atypia despite conventional chemotherapeutic intervention. In the 5-FU-pSL group, while there was an observable reduction in cellular invasion, pleomorphic nuclei remained evident in tumor cells that continued to infiltrate the surrounding adipose tissue matrix. This suggests partial therapeutic response while maintaining some characteristics of malignant transformation. Significantly, the 5-FU-iRGD-FA-pSL treatment group demonstrated remarkable therapeutic efficacy, characterized by the restoration of normal cellular architecture. The tissue sections exhibited histological features consistent with healthy breast tissue, indicating substantial regression of malignant characteristics and successful tissue remodelling.

Fig. 8 Hematoxylin and eosin (H&E) staining was conducted on tissues from DMBA-induced rats, with digital image analysis performed for further evaluation (scale bar: 50 μm). Three-dimensional (3D) image reconstruction and analysis were carried out using ImageJ software (NIH). A dataset of the stained regions was created through thresholding, followed by analysis of pixel intensity. To quantify and assess the staining patterns, 3D interactive surface plots and log-histogram analysis were applied.

3.8.6 Biosafety evaluation of 5-FU-pSL formulations. One of the major challenges hindering the widespread clinical application of nanoformulation-based anticancer therapies is the potential toxicity to vital organs. To assess the safety profile of the 5-FU-pSL and 5-FU-iRGD-FA-pSL formulations, histopathological analysis was conducted to evaluate any potential harm to key organs, including the liver, kidneys, lungs, and spleen (Fig. 9). Histological examination of these organs from animals treated with 5-FU-pSL, and 5-FU-iRGD-FA-pSL revealed no significant histopathological alterations. Microscopic images demonstrated that the major organs appeared unaffected, showing no signs of inflammation, necrosis, or other forms of tissue damage. These findings suggest that both 5-FU-pSL and 5-FU-iRGD-FA-pSL formulations are well-tolerated at therapeutic doses, exhibiting excellent biocompatibility and minimal toxicity to vital organs.

Fig. 9 Histopathological study of the liver, kidney, lungs, spleen and heart from female Wistar rats treated with pure 5-FU and 5-FU-loaded 5-FU-pSL formulations compared to control. Tissue sections were stained with hematoxylin and counterstained with eosin, and histopathological analyses were conducted to evaluate potential tissue toxicity. Images were captured using a 400× objective lens with a scale bar of 50 μm.

3.8.7 Anti-inflammatory response. The therapeutic efficacy of various 5-FU formulations was evaluated through a comprehensive analysis of inflammatory markers and matrix metalloproteinases (Fig. 10). The DMBA-induced toxic group exhibited significant elevation in pro-inflammatory cytokines, serving as a positive control for inflammation-mediated carcinogenesis.

Fig. 10 Estimation of inflammatory cytokines after treatment with different 5-FU-pSL formulations. M ± SD (n = 3) [n.s. (not significant) * < 0.05, ** < 0.01, *** < 0.001 (in comparison to toxic control)].

Matrix metalloproteinase-1 (MMP1) levels in the toxic group (310.5 ± 25.3 pg mL⁻¹) were markedly elevated compared to the control (95.2 ± 8.4 pg mL⁻¹). Treatment with free 5-FU moderately reduced MMP1 expression (280.5 ± 18.6 pg mL⁻¹, p < 0.05), while 5-FU-pSL showed enhanced suppression (210.4 ± 22.8 pg mL⁻¹, p < 0.01). The 5-FU-iRGD-FA-pSL formulation demonstrated superior efficacy in reducing MMP1 levels (82.5 ± 12.7 pg mL⁻¹, p < 0.001), approaching control values. Tumor Necrosis Factor-alpha (TNF-α) expression showed the most dramatic variation among cytokines. The toxic group exhibited substantially elevated levels (430.5 ± 28.4 pg mL⁻¹) compared to control (18.5 ± 3.2 pg mL⁻¹). The sequential improvement in TNF-α suppression was observed across treatment groups: free 5-FU (350.2 ± 12.6 pg mL⁻¹, p < 0.05), 5-FU-pSL (280.4 ± 8.5 pg mL⁻¹, p < 0.01), and 5-FU-iRGD-FA-pSL (48.6 ± 12.3 pg mL⁻¹, p < 0.001). Interleukin-6 (IL-6) levels demonstrated a similar trend, with the toxic group showing significant elevation (210.5 ± 12.4 pg mL⁻¹) versus control (28.4 ± 2.1 pg mL⁻¹). The 5-FU-iRGD-FA-pSL group exhibited superior IL-6 suppression (72.5 ± 8.4 pg mL⁻¹, p < 0.001) compared to free 5-FU (170.2 ± 8.5 pg mL⁻¹, p < 0.05) and 5-FU-pSL (125.4 ± 5.2 pg mL⁻¹, p < 0.01). Similarly, Interleukin-1β (IL-1β) levels in the toxic group (310.2 ± 32.5 pg mL⁻¹) were significantly reduced by 5-FU-iRGD-FA-pSL treatment (108.4 ± 8.6 pg mL⁻¹, p < 0.001), showing superior efficacy compared to both free 5-FU (210.5 ± 8.4 pg mL⁻¹, p < 0.05) and 5-FU-pSL (185.2 ± 12.4 pg mL⁻¹, p < 0.01).

The cytokine profile analysis demonstrates a clear hierarchical efficacy pattern: 5-FU-iRGD-FA-pSL > 5-FU-pSL > free 5-FU, suggesting that the dual-targeted liposomal formulation significantly enhances the anti-inflammatory and anti-tumor effects of 5-FU. Statistical significance was observed across all treatment groups (p < 0.05), with 5-FU-iRGD-FA-pSL consistently showing the most potent suppression of inflammatory markers (p < 0.001). These findings indicate that the enhanced delivery system improves the cytotoxic effects of 5-FU and significantly modulates the inflammatory microenvironment associated with tumor progression. The superior efficacy of 5-FU-iRGD-FA-pSL can be attributed to its dual-targeting mechanism, facilitating improved drug delivery to tumor cells while effectively suppressing inflammatory cascades.

4. Conclusion

The study successfully demonstrates the development and application of a dual-targeting, pSL system for delivering 5-FU in BC therapy. The 5-FU-iRGD-FA-pSL formulation showed enhanced targeting efficiency through receptor-mediated endocytosis and improved drug penetration via the iRGD peptide. In vitro and in vivo results indicate that this formulation significantly enhances the cytotoxic effects of 5-FU against BC cells while reducing systemic side effects. The dual-targeting strategy, combined with pH-sensitive drug release, offers a promising approach to overcome the limitations of conventional chemotherapy. This innovative delivery system has the potential to improve therapeutic outcomes for BC patients, paving the way for more effective and safer cancer treatment options.

Author contributions

Prashant Pandey: conceptualization, data curation, formal analysis, methodology, Dilip Kumar Arya: formal analysis, methodology, Anit Kumar: software, writing – review and editing. Ajeet Kumar: visualization, validation, writing – review and editing. Yogendra Kumar Mishra: visualization, validation, writing – review and editing. P. S. Rajinikanth: conceptualization, resources, project administration, supervision. All authors have contributed to the original draft writing and revision of the manuscript.

Data availability

All relevant data are within the paper.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The funding was received by Prashant Pandey and funded by the Anusandhan National Research Foundation (ANRF) formerly known as Science and Engineering Research Board (SERB), New Delhi, India, under Overseas Visiting Doctoral Fellowship (OVDF), Fellowship ID: SB/S9/Z-16/2016-IV (2022). The authors also thank Lipoid GmbH, Ludwigshafen, Germany, for providing gift samples.

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