PEGylated Liposomes Enhance Bufalin’s Solubility, Pharmacokinetics, and Glioma Cytotoxicity

Abstract

Bufalin, a cardiac glycoside extracted from Venenum Bufonis, exhibits notable cardiotonic, antiviral, immune‑modulatory, and antitumor activities. However, its hydrophobic nature limits systemic delivery. In this study we formulated bufalin into PEGylated liposomes using FDA‑approved excipients and compared their physicochemical properties, in vitro efficacy, and in vivo pharmacokinetics with non‑PEGylated liposomes and free drug. Both liposomal systems displayed homogeneous particle sizes (127.6 ± 3.6 nm for non‑PEGylated, 155.0 ± 8.5 nm for PEGylated), high entrapment efficiencies (> 76 %), and distinct surface charges (2.2 mV vs. –18.0 mV). The PEGylated formulation released bufalin more slowly in vitro and, in Sprague‑Dawley rats, prolonged plasma half‑life (87.8 min vs. 40.5 min for free drug) and increased AUC by 5.5‑fold. Cytotoxicity assays on U251 glioma cells revealed superior potency of PEGylated liposomes compared to free bufalin. These results demonstrate that PEGylated liposomes markedly improve bufalin’s aqueous solubility, bioavailability, and antitumor efficacy, supporting their potential as a glioma drug delivery platform.

Background

Globally, cancer incidence is rising, with gliomas posing a particularly lethal challenge due to the blood‑brain barrier (BBB) and multidrug resistance [1,2]. Bufalin, isolated from the skin and parotid secretions of toads such as Bufo bufo gargarizans and Bufo melanostictus, has been reported to possess cardiotonic, antiviral, immunoregulatory, and antitumor properties [3–7]. Its clinical translation is hindered by poor aqueous solubility and rapid clearance [8]. Liposomal encapsulation can enhance solubility, protect drugs from degradation, and facilitate BBB penetration [9–10]. Yet, conventional liposomes are rapidly opsonized and cleared by the mononuclear phagocyte system. Surface PEGylation imparts a steric shield, extending circulation time and improving pharmacokinetics [11]. No comprehensive study has yet compared the pharmacokinetics of free bufalin, non‑PEGylated, and PEGylated liposomal formulations following intravenous delivery. This work addresses that gap by developing PEGylated liposomes for bufalin and evaluating their physicochemical characteristics, in vitro release, cytotoxicity, and in vivo pharmacokinetics in rats.

Methods

Chemicals and Reagents

Bufalin (≥ 98 % purity) was sourced from BaoJi Chenguang Technology Development Co., Ltd. Lipid components—L‑α‑phosphatidylcholine, cholesterol, and DSPE‑PEG2000—were purchased from Sigma‑Aldrich. All solvents were HPLC grade, and MilliQ water was used throughout.

Animals and Cell Lines

Male Sprague‑Dawley rats (250 ± 20 g) were maintained under standard conditions. Human tumor lines (SW620, PC‑3, MDA‑MB‑231, A549, U251, U87, HepG2) were cultured in RPMI‑1640 supplemented with 10 % FBS and antibiotics at 37 °C, 5 % CO₂.

Liposome Preparation

Bufalin was incorporated into liposomes via thin‑film rehydration followed by high‑pressure homogenization (500 bar, 35 °C, 10 cycles). Non‑PEGylated liposomes contained bufalin, cholesterol, and L‑α‑phosphatidylcholine (10:30:60 mol %) whereas PEGylated liposomes added DSPE‑PEG2000 (5 mol %) to yield a 10:30:55:5 ratio. The resultant suspension was extruded twice through 0.2 µm polycarbonate membranes to produce unilamellar vesicles.

Characterization

Size, Zeta Potential, and Morphology

Dynamic light scattering (DLS) measured hydrodynamic diameters and zeta potentials (Delsa™ Nano, Beckman Coulter). TEM (Hitachi H‑7650, 200 kV) confirmed spherical morphology.

Entrapment Efficiency

Encapsulation was quantified by HPLC on a SinoChrom ODS‑BP C18 column (250 × 4.6 mm, 5 µm). A gradient of acetonitrile and 0.1 % KH₂PO₄ (pH 3.8) at 1.0 mL min⁻¹ was employed. Entrapment (%) = (W₁/W₂) × 100, where W₁ is bufalin recovered from the liposome core and W₂ the total added dose.

Stability Assessment

Samples stored at 4 °C were evaluated on days 0, 7, 15, 30, and 90 for particle size, zeta potential, and leakage (W₀ – Wₓ)/W₀ × 100 %.

In Vitro Release

Dialysis bags (MWCO 30 kDa) containing 1 mL of liposome suspension were immersed in 50 mL PBS (pH 7.4) with 10 % FCS at 37 °C. Samples were collected at specified time points and analyzed by HPLC.

Cytotoxicity

MTT assays were performed on U251 and U87 glioma cells exposed to blank, PEGylated, or bufalin‑loaded liposomes. Cell viability was read at 570 nm after 24 h incubation.

In Vivo Pharmacokinetics

Rats received a 0.5 mg kg⁻¹ IV dose of free bufalin, non‑PEGylated liposomes, or PEGylated liposomes. Blood samples were collected up to 10 h post‑dose, centrifuged, and plasma stored at –20 °C. Bufalin concentrations were quantified by liquid‑liquid extraction followed by HPLC, and non‑compartmental analysis yielded C_max, T½, and AUC.

Statistical Analysis

Data are mean ± SD (n = 3). One‑way ANOVA (GraphPad Prism) determined significance at p < 0.05.

Results

Physicochemical Properties

Both liposomal preparations exhibited uniform size distributions (<200 nm) and high encapsulation (76–78 %). The PEGylated vesicles possessed a more negative surface charge (–18 mV vs. +2 mV), indicative of enhanced colloidal stability (Fig. 1).

Stability

Over 90 days at 4 °C, PEGylated liposomes showed minimal size increase (≤ 8 %) and leakage (< 18 %) compared to non‑PEGylated liposomes (leakage up to 31 %).

Release Profile

Free bufalin dissolved rapidly in PBS/FCS, while PEGylated liposomes released the drug at a slower, sustained rate (Fig. 2). The lag in release correlates with the steric barrier imposed by DSPE‑PEG2000.

Cytotoxicity

Across tumor lines, bufalin inhibited growth in a dose‑dependent manner, with IC₅₀ values lowest for U251 and U87 glioma cells (Table S2). PEGylated liposomes reduced U251 viability more effectively than free bufalin or non‑PEGylated liposomes, while blank formulations showed no cytotoxicity (Fig. 3).

Pharmacokinetics

In rats, PEGylated liposomes extended plasma half‑life (87.8 min) and AUC (139 µg min mL⁻¹) relative to free bufalin (40.5 min, 25 µg min mL⁻¹). Non‑PEGylated liposomes also improved pharmacokinetics (54.4 min, 58 µg min mL⁻¹) but to a lesser extent (Fig. 4).

Discussion

Our PEGylated formulation achieved a particle size within the optimal 100–200 nm window for tumor penetration, while the negative surface charge conferred long‑term stability. The sustained release profile aligns with the known barrier effect of PEG, which protects liposomal contents from premature leakage. Cytotoxicity assays confirm that encapsulation does not compromise, and may even enhance, bufalin’s antitumor potency against glioma cells—likely due to improved cellular uptake and controlled intracellular release.

Pharmacokinetic data underscore the clinical relevance: PEGylation doubled the plasma residence time and more than quintupled systemic exposure, potentially translating into higher tumor accumulation via the enhanced permeability and retention (EPR) effect. These findings are consistent with previous reports on PEG‑modified liposomal drugs that achieved prolonged circulation and reduced clearance [12–14].

While the study demonstrates clear advantages of PEGylated liposomes, further investigations are warranted to map tissue distribution, assess BBB translocation, and evaluate therapeutic efficacy in orthotopic glioma models. Nonetheless, the data support PEGylated liposomes as a promising delivery platform for bufalin in cancer therapy.

Conclusions

PEGylated liposomes markedly improve bufalin’s solubility, extend its plasma half‑life, and enhance glioma cell cytotoxicity. This nanocarrier system offers a viable strategy for delivering bufalin to the brain and warrants further preclinical development.