Bufalin-Loaded PEGylated Liposomes: Enhanced Antitumor Efficacy, Reduced Toxicity, and Optimized Brain Delivery

Abstract

Bufalin, a cardiac glycoside extracted from Venenum Bufonis, demonstrates strong antitumor activity but is limited by poor bioavailability and significant systemic toxicity when administered as a free drug. In this study we evaluated bufalin‑loaded PEGylated liposomes (BF/PEG‑LP) in terms of physicochemical characteristics, safety, in‑vitro cytotoxicity, in‑vivo antitumor activity, acute toxicity, and tissue distribution. Red‑blood‑cell hemolysis assays showed that BF/PEG‑LP caused markedly less hemolysis than free bufalin. Blank liposomes were non‑toxic to HepG2 and HCT116 cells. BF/PEG‑LP induced dose‑dependent apoptosis in HepG2, HCT116, A549, and U251 cells with IC₅₀ values of 21.40 ± 2.39, 21.00 ± 3.34, 43.39 ± 6.43, and 31.14 ± 2.58 ng/mL, respectively. In U251 xenografts, BF/PEG‑LP significantly suppressed tumor growth, achieving a 38.7 % inhibition rate at 2 mg/kg versus 27.6 % for free bufalin. Pharmacological evaluation revealed transient behavioral changes at the highest dose, while the median lethal concentration (LD₅₀) increased from 0.156 mg/kg (free bufalin) to 3.03 mg/kg (BF/PEG‑LP). Tissue‑distribution studies demonstrated a 20 % higher brain concentration and a 30 % lower heart concentration for BF/PEG‑LP compared with free bufalin. These findings confirm that PEGylation improves bufalin’s pharmacokinetics, reduces systemic toxicity, and enhances brain exposure, supporting its further development as a glioma therapeutic.

Introduction

Bufalin (BF) is a triterpenoid lactone that exhibits anti‑inflammatory, cardiotonic, antiviral, and antitumor effects. Preclinical work has shown BF’s efficacy against hepatocellular carcinoma, melanoma, colorectal carcinoma, glioma, and ovarian carcinoma, primarily through inhibition of cell proliferation and induction of apoptosis. However, clinical translation has been hampered by BF’s low aqueous solubility, limited oral bioavailability, and dose‑dependent toxicity to normal tissues. Liposomal encapsulation offers a proven strategy to enhance drug solubility, protect the active agent from premature degradation, and facilitate targeted delivery across the blood‑brain barrier (BBB). PEGylation of liposome surfaces further extends circulation time and reduces uptake by the mononuclear phagocytic system. In earlier work we prepared BF/PEG‑LP and characterized their size, zeta potential, and entrapment efficiency. The current study extends that work by investigating BF/PEG‑LP’s safety profile, antitumor potency, acute toxicity, and biodistribution in preclinical models.

Materials and Methods

Reagents

BF and cinobufagin (CBG) were purchased from BaoJi Chenguang Technology Development Co., Ltd. (98 % purity). Doxorubicin (DOX) served as a positive control. BF/PEG‑LP were prepared in-house, yielding an average diameter of 155 ± 8 nm, zeta potential of –18.5 ± 4.5 mV, and entrapment efficiency of 76.3 ± 4.2 %. All other chemicals were analytical grade.

Animals

Male Sprague‑Dawley rats, Kunming mice, and 6‑week‑old nude Balb/c mice were used. Animals were acclimated, fasted overnight, and all protocols were approved by the Institutional Animal Care and Use Committee (Approval 2017‑0603‑R).

Hemolysis Assay

RBCs from rats were incubated with BF or BF/PEG‑LP, and hemolysis was quantified spectrophotometrically at 398 nm. The hemolysis rate was calculated relative to positive (water) and negative (saline) controls.

Cytotoxicity of Blank Liposomes

Blank liposomes were tested on HepG2 and HCT116 cells using the CCK‑8 assay. Cell viability remained >95 % at concentrations up to 200 µg/mL, confirming their non‑toxic nature.

In‑Vitro Antitumor Activity

HepG2, HCT116, A549, and U251 cells were exposed to serial dilutions of BF/PEG‑LP for 24 h. Cell viability was assessed by CCK‑8, and IC₅₀ values were derived from dose–response curves.

U251 Xenograft Model

U251 cells (1 × 10⁷) were subcutaneously injected into nude mice. Once tumors reached ~50 mm³, mice received i.p. injections of BF or BF/PEG‑LP at 0.5, 1.0, or 2.0 mg/kg daily for 21 days. Tumor volume, body weight, and behavioral observations were recorded. Tumors were harvested, weighed, and histologically examined by H&E staining.

Acute Toxicity

Kunming mice received a single i.v. dose of BF or BF/PEG‑LP (1.0, 0.37, 0.14, 0.05, or 0.02 mg/kg for BF; 4.0, 2.83, 2.0, 1.41, or 1.0 mg/kg for BF/PEG‑LP). Survival was monitored over 14 days, and the LD₅₀ was calculated using the Reed–Muench method.

Tissue Distribution (HPLC)

Sprague‑Dawley rats received i.p. 0.5 mg/kg BF or BF/PEG‑LP. Heart, liver, spleen, lung, kidney, and brain tissues were collected at 5, 15, 45, and 90 min. Samples were homogenized, extracted with ethyl acetate, and analyzed by HPLC (Waters 2995/2996) using a mobile phase of acetonitrile:0.1 % formic acid/water (65:35, v/v). Calibration curves (20–2000 ng/mL) exhibited R² > 0.9977.

Results and Discussion

Physicochemical Properties and Safety

TEM imaging confirmed a uniform spherical morphology. FT‑IR spectra displayed characteristic BF peaks, confirming successful encapsulation. The hemolysis rate of BF/PEG‑LP was <0.5 % at concentrations up to 200 µg/mL, a 95 % reduction compared with free BF (p < 0.01). Blank liposomes showed no cytotoxicity in HepG2 and HCT116 cells.

Stability Across pH

BF/PEG‑LP maintained integrity in PBS at pH 7.4, whereas a pH 5.0 buffer induced a significant increase in absorbance, indicating reduced stability under acidic conditions.

In‑Vitro Cytotoxicity

BF/PEG‑LP inhibited cell viability in a dose‑dependent manner across all tested lines. IC₅₀ values were 21.4 ± 2.4, 21.0 ± 3.3, 43.4 ± 6.4, and 31.1 ± 2.6 ng/mL for HepG2, HCT116, A549, and U251, respectively. The activity surpassed that of DOX (50 µg/mL) at concentrations >40 ng/mL. Sensitivity ranking: HCT116 > HepG2 > U251 > A549.

In Vivo Antitumor Efficacy

BF/PEG‑LP significantly reduced tumor volume and weight compared with free BF and vehicle. At 2 mg/kg, BF/PEG‑LP achieved a 38.7 % inhibition rate versus 27.6 % for free BF. H&E staining revealed extensive necrosis in BF/PEG‑LP‑treated tumors, proportional to dose.

Behavioral and Pharmacological Effects

High‑dose BF/PEG‑LP induced mild tremors and salivation; locomotor activity was modestly decreased relative to controls (p < 0.05). Rod‑climbing tests showed improved coordination after one week of treatment (p < 0.01).

Acute Toxicity

LD₅₀ values increased from 0.156 mg/kg (free BF) to 3.03 mg/kg (BF/PEG‑LP), reflecting a ~19‑fold reduction in acute toxicity. Histopathology revealed cardiac myocyte disruption and hepatic necrosis in free‑BF‑treated mice but minimal changes in BF/PEG‑LP groups.

Tissue Distribution

BF/PEG‑LP yielded higher brain concentrations (333 ± 92.5 ng/g at 5 min) and lower heart concentrations (95.0 ± 18.5 ng/g at 5 min) compared with free BF. Liver accumulation was modestly increased (187 ± 31.0 ng/g vs. 163 ± 35.0 ng/g). Brain levels remained detectable up to 90 min, indicating prolonged residence.

Conclusion

PEGylated liposomal encapsulation of bufalin markedly improves its pharmacokinetic profile, reduces systemic and cardiac toxicity, and enhances brain delivery. BF/PEG‑LP demonstrates potent antitumor activity in vitro and in vivo, particularly against glioma, and offers a promising platform for clinical translation. Future studies should investigate the accelerated blood clearance phenomenon and optimize dosing schedules for maximal therapeutic benefit.

Availability of Data and Materials

All data generated or analyzed during this study are included in the article and its supplementary information files.