Optimizing Daidzein-Loaded Long‑Circulating Liposomes: Enhanced Oral Bioavailability and Prolonged Release

Background

Daidzein is an isoflavone that appears exclusively in soybeans and related legumes. Its therapeutic profile includes cardiovascular protection, menopausal symptom relief, osteoporosis prevention, reduction of hormone‑related cancer risk, and anti‑inflammatory activity. However, its dual poor hydrophilicity and lipophilicity, coupled with rapid glucuronidation and sulfation in the gut, limit its oral bioavailability. Recent strategies—phospholipid complexes, self‑assembled micelles, and PLGA nanoparticles—have attempted to overcome these limitations, but each has drawbacks in stability or drug loading.

Liposomes, as versatile drug carriers, encapsulate both hydrophilic and hydrophobic agents, enhancing intestinal permeability, protecting against enzymatic degradation, and reducing off‑target toxicity. Conventional liposomes, however, are rapidly cleared by the mononuclear phagocyte system (MPS). Surface modification with hydrophilic polymers such as polyethylene glycol (PEG) generates long‑circulating liposomes that resist opsonization and extend systemic circulation, thereby improving bioavailability. This study investigates the preparation of a PEG‑modified liposome carrying daidzein, its physicochemical properties, in‑vitro release kinetics, and in‑vivo pharmacokinetics in rats, providing a foundation for clinical translation.

Methods

Materials

SPC (Lipoid GmbH, Germany), cholesterol and DSPE‑mPEG2000 (AVT Pharmaceutical Co., Shanghai), daidzein (≥ 98 % purity, Yuanye Biotechnology, Shanghai), apigenin (internal standard, Delge Pharmaceutical Technology, Nanjing), HPLC‑grade methanol and acetonitrile (TEDIA, USA), chloroform and methanol (Sinopharm, Shanghai), Milli‑Q water, and other reagents were used as received. All solvents were of analytical grade.

Animals

Ten male Sprague‑Dawley rats (200–210 g) were sourced from the Hubei Province Animal Center (license SCXK (E) 2017–0012) and housed under standard laboratory conditions. The study protocol received approval from the Ethics Committee of Hubei University and adhered to the NIH Guide for the Care and Use of Laboratory Animals.

Preparation of Daidzein Long‑Circulating Nanoliposome (DLCL)

An orthogonal design (four factors, three levels) evaluated the impact of SPC‑cholesterol ratio (A), drug‑to‑lipid ratio (B), hydration temperature (C), and ultrasonic time (D) while maintaining 5 % DSPE‑mPEG2000. The optimal parameters were SPC : cholesterol = 55 : 40 (molar), drug‑to‑lipid = 1 : 10 (w/w), hydration at 50 °C, and 24 min sonication. The liposomes were formed by dissolving SPC, cholesterol, DSPE‑mPEG2000, and daidzein in a 1:4 (v/v) chloroform‑methanol mixture, evaporating under vacuum at 40 °C to a thin film, hydrating with 20 mL ultrapure water in an ice bath while sonicated at 80 W, and extruding sequentially through 0.45 µm and 0.22 µm membranes. The final suspension was stored at 4 °C; for lyophilization, 3 % sucrose was added as a cryoprotectant.

Determination of Daidzein in DLCL by HPLC

A Phenomenex ODS column (150 × 4.6 mm, 5 µm) at 40 °C was employed with a gradient of 10 mM ammonium acetate (A) and methanol (B). Flow rate: 1 mL min⁻¹; detection at 240 nm; injection volume 10 µL. Linear range: 0.313–50 µg mL⁻¹ (R² = 0.9999). Retention time: 4.30 min. Method validation confirmed accuracy, precision, and stability (Table 1).

Particle Size and Morphology

The DLCL size and zeta potential were measured by dynamic light scattering (Zetasizer Nano90). TEM imaging (JEM‑2100) confirmed a uniform, spherical bilayer structure. The mean diameter was 156.1 ± 3.0 nm with a polydispersity index (PDI) of 0.294 ± 0.012 and a zeta potential of –49 ± 0.6 mV.

Encapsulation Efficiency and Drug Loading

EE and drug loading were quantified via dialysis: 500 µL of DLCL was placed in a 8–14 kDa MWCO bag, dialyzed against 20 mL water for 12 h, and free drug concentration (C₁) measured. Total drug (C₀) was obtained by methanol extraction. EE (%) = (C₀ – C₁)/C₀ × 100; drug loading (%) = C₀ × V₀ × EE/W₀ × 100. The optimized DLCL yielded EE = 85.3 ± 3.6 % and loading = 8.2 ± 1.4 %.

In‑Vitro Drug Release

Release was assessed in simulated gastric fluid (pH 1.2, 0.1 M HCl, 0.5 % Tween‑80) and intestinal fluid (pH 6.9, 25 mM PBS, 0.5 % Tween‑80) using a dialysis bag (8–14 kDa). Samples were withdrawn at 0–144 h, replenished, and daidzein quantified by HPLC. DLCL extended cumulative release to 48 h at both pH levels, whereas free drug reached completion within 12–24 h.

Pharmacokinetics in Rats

Rats were fasted overnight and orally dosed with either free daidzein (30 mg kg⁻¹) or DLCL (30 mg kg⁻¹). Blood was collected at 0.5–36 h, processed to plasma, and analyzed by LC‑MS/MS (GL Inertsustain C18 column, 40 °C, 0.2 mL min⁻¹). The assay monitored the m/z 253.0 → 224.15 transition for daidzein and 269.0 → 117.05 for apigenin (internal standard). Non‑compartmental analysis (DAS3.0) yielded MRT, t½, and AUC₀–t values.

Results and Discussion

Optimized Nanoliposome Characteristics

The orthogonal array identified drug‑to‑lipid ratio and SPC‑cholesterol ratio as the primary determinants of size, with minimal effect on EE. The final formulation produced a narrow size distribution, excellent encapsulation, and robust negative surface charge, indicating stable colloidal behavior. TEM images corroborated a well‑formed bilayer, and the inclusion of daidzein did not disrupt liposomal architecture.

In‑Vitro Release Profile

Daidzein released from DLCL exhibited a sustained profile: 18 % at 1 h and 100 % by 48 h in gastric fluid; 3 % at 1 h and 100 % by 48 h in intestinal fluid. In contrast, free daidzein released 85 % and 73 % at 1 h in gastric and intestinal media, respectively, achieving full release within 12–24 h. The slower release at lower pH is attributed to acid‑mediated destabilization of the lipid bilayer.

In‑Vivo Pharmacokinetics

DLCL administration produced a markedly higher plasma concentration curve compared with free drug. Key pharmacokinetic parameters: AUC₀–t = 1515.5 ± 532.4 µg L⁻¹ h (2.5‑fold increase), MRT = 1.6‑fold, and t½ = 1.8‑fold relative to free daidzein. The extended MRT and t½ reflect reduced first‑pass metabolism and prolonged systemic exposure, confirming the therapeutic advantage of the PEG‑modified liposomal delivery.

Conclusions

DLCL, prepared with 85.3 % encapsulation efficiency and 8.2 % drug loading, demonstrates superior in‑vitro release kinetics and significantly enhances oral bioavailability in rats. The formulation achieves a 2.5‑fold AUC increase and prolongs systemic exposure by 1.6‑ to 1.8‑fold, underscoring its potential as a clinically viable oral delivery system for daidzein.

Availability of Data and Materials

All data, materials, and protocols are available to qualified researchers without undue restriction.

Abbreviations

AUC

Area under the time‑concentration curve;

DLCL

Daidzein long‑circulating liposomes;

DSPE‑mPEG2000

Distearoyl phosphoethanolamine‑PEG2000;

EE

Encapsulation efficiency;

HPLC

High‑performance liquid chromatography;

LC‑MS/MS

Liquid chromatography‑tandem mass spectrometry;

MRT

Mean residence time;

t½

Elimination half‑life;