Optimizing Glycyrrhetinic Acid Liposomes via Lyophilization Monophase Solution: Preformulation, Design, and In‑Vitro Evaluation

Abstract

In this study, glycyrrhetinic acid (GA) liposomes were successfully formulated using the lyophilization monophase solution method. Preformulation work examined the solubility of soybean phosphatidylcholine (SPC), cholesterol, and GA in tert‑butyl alcohol/water (TBA/water) co‑solvents. We investigated how TBA volume percentage influences sublimation rate, and characterized the lyophilized GA by DSC, XRD, and FTIR. XRD indicated an amorphous state for GA, while FTIR confirmed that no chemical changes occurred. Solubility tests revealed a marked increase in aqueous GA solubility. Using a Box‑Benhnken design and lyoprotectant selection, we identified the optimal formulation—508 mg SPC, 151 mg cholesterol, 55 % TBA, and a 4:1 trehalose/SPC weight ratio. Under these conditions, reconstituted liposomes exhibited an encapsulation efficiency of 74.87 % and a mean diameter of 191 nm. In‑vitro release studies showed sustained release in two media, and cellular uptake assays demonstrated a time‑dependent uptake by Hep G2 cells.

Background

Glycyrrhetinic acid (GA), a triterpene saponin extracted from Glycyrrhiza roots, exhibits antimicrobial, antiviral, and anticancer activity and is clinically used for chronic hepatitis and liver cancer. According to the Biopharmaceutical Classification System, GA is a class II drug; its low polarity, high hydrophobicity, and poor solubility limit oral bioavailability and can cause dose‑dependent sodium retention and potassium loss, leading to hypertension. Enhancing absorption and maintaining therapeutic concentrations through targeted formulation is therefore essential.

Liposomes are widely recognized for their biocompatibility, ability to target lymph nodes and tumor sites via the enhanced permeability and retention effect, and versatility in carrying both hydrophobic and hydrophilic drugs. However, conventional aqueous liposome suspensions are unstable, prone to leakage, fusion, and phospholipid hydrolysis, which hampers long‑term storage. Proliposomes—dry, fluid powders that reconstitute into liposomes upon hydration—offer a solution. Traditional proliposome production by spray‑drying or freeze‑drying has drawbacks: spray‑drying can damage thermosensitive drugs, and freeze‑drying is time‑consuming and costly due to prolonged water removal.

A recent innovation, the lyophilization monophase solution method, dissolves lipids, drug, and a water‑soluble lyoprotectant in a tert‑butyl alcohol/water co‑solvent, then freeze‑drying produces a dry powder that spontaneously forms liposomes when water is added. Advantages include rapid sublimation, one‑step processing suitable for large‑scale production, low residual solvent risk (TBA is class 3), sterile powder output, and compatibility with poorly water‑soluble or unstable drugs.

Despite these benefits, knowledge gaps remain: the impact of TBA/water concentration on sublimation rate, solid‑state changes of the drug post‑lyophilization, and the hydration‑assembly process of the powder. This study uses GA as a model to address these questions, optimizing formulation and process variables via Box‑Benhnken design, evaluating lyoprotectant effects, and assessing in‑vitro release and cellular uptake.

Methods / Experimental

Materials

Glycyrrhetinic acid (≥ 98 % purity) was sourced from Dalian Meilun Biology Technology Co., Ltd. Soybean phosphatidylcholine (Lipoid S100) and cholesterol were obtained from Lipoid GmbH and J&K Scientific Ltd., respectively. FITC‑PEG‑DSPE (MW 2000) was from Shanghai Ponsure Biotech, Inc. Tert‑butyl alcohol (≥ 98 %) and all other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Deionized water was produced by a Milli‑Q system.

Solubility Studies

Saturated solutions of GA, SPC, and cholesterol were prepared in TBA/water mixtures at temperatures ranging from 25 °C to 45 °C. GA solubility was quantified by HPLC using a C18 column (4.6 × 250 mm, 5 µm) and a methanol/water (90:10 v/v) mobile phase at 1.0 ml min⁻¹, detecting at 250 nm. SPC and cholesterol solubility was estimated via a turbidimetric method, monitoring absorbance at 655 nm.

Lyophilization Monophase Solution Preparation

GA, SPC, and cholesterol were dissolved in TBA at 45 °C; a selected lyoprotectant (mannitol, lactose, sucrose, or trehalose) was dissolved in water at 45 °C. The two solutions were mixed to yield an isotropic monophase solution (60 ml). After filtration (0.22 µm), 2.0 ml aliquots were placed in 10 ml freeze‑drying vials, pre‑frozen at − 40 °C for 12 h, and lyophilized at − 50 °C for 24 h (chamber pressure 1–20 Pa).

Particle Size and Encapsulation Efficiency

Reconstituted liposomes were prepared by dispersing 5 mg of proliposome powder in 5 ml purified water, vortexing for 1 min, repeating after 15 min. Size was measured by dynamic light scattering (Nano ZS90, Malvern). Encapsulation efficiency (EE) was determined by ultrafiltration‑centrifugation: 1 ml of dispersion was filtered through a 50 kDa MWCO filter, the filtrate quantified by HPLC, and EE calculated via EE = (1 − W_free/W_total) × 100 %.

Sublimation Rate and Vapor Pressure

TBA/water mixtures of varying TBA volume percentages (10–90 %) were pre‑frozen and lyophilized; the time until complete sublimation was recorded to calculate rate (µl min⁻¹). Vapor pressure was measured using a static ebulliometer and the Antoine equation.

Physicochemical Characterization

DSC (HSC‑1, 25–350 °C, 10 °C min⁻¹, N₂ atmosphere) assessed thermal transitions. XRD (D8 Focus, Cu‑Kα, 5°–60°, 5° min⁻¹) evaluated crystallinity. FTIR (Nicolet 6700, 4000–400 cm⁻¹, 4 cm⁻¹ resolution) verified chemical integrity.

Stability

Proliposome powders were stored at 25 °C, nitrogen‑filled, in the dark. After 6 months, EE and particle size of reconstituted liposomes were reassessed.

In‑Vitro Release

Release was studied by dialysis (MWCO 8000–14000 Da) in PBS (pH 7.4) or 0.1 % Tween 80 saline at 37 °C, stirring at 300 rpm. Samples were withdrawn up to 12 h, replaced with fresh medium, and GA quantified by HPLC.

Cellular Uptake

FITC‑labeled GA liposomes were incubated with HepG2 cells (1 × 10⁴ cells mL⁻¹) for 0.5, 1, 2, and 4 h. After washing and trypan blue quenching, cells were lysed with Triton X‑100. Fluorescence (excitation 495 nm, emission 520 nm) was measured and normalized to protein content (BCA assay).

Results and Discussion

Solubility and Sublimation

GA solubility in TBA/water rose progressively with both TBA volume percentage and temperature; solubility exceeded 0.5 mg mL⁻¹ when TBA > 40 %. SPC and cholesterol required decreasing TBA volumes as temperature increased above 35 °C, confirming the favorable dissolution effect of TBA.

Sublimation rate increased from 4 µl min⁻¹ at 10 % TBA to over 10 µl min⁻¹ above 60 % TBA, attributed to higher saturated vapor pressure and finer needle‑shaped ice crystals observed in optical microscopy.

Physicochemical Changes Post‑Lyophilization

DSC showed a shift of the GA melting peak to lower temperatures as TBA content decreased, while XRD confirmed complete loss of crystalline peaks, indicating an amorphous state. FTIR spectra remained unchanged, proving chemical stability. Amorphization enhanced aqueous solubility from 6.36 µg mL⁻¹ (raw) to 19–64 µg mL⁻¹ depending on TBA ratio.

Single‑Factor Screening

Optimal SPC (480 mg), cholesterol (120 mg), and TBA (50 %) were identified based on highest combined score of EE and mean diameter.

Box‑Benhnken Optimization

Using SPC, cholesterol, and TBA as independent variables, the model predicted optimal conditions: 508 mg SPC, 151 mg cholesterol, 55 % TBA, yielding EE = 68.55 % and mean diameter = 220 nm. Interaction plots confirmed synergistic effects.

Lyoprotectant Selection

Trehalose outperformed lactose, sucrose, and mannitol. A trehalose/SPC ratio of 4:1 maximized EE (74.87 %) and minimized particle size (191 nm), with superior protection during freeze‑thaw cycles.

Transmission Electron Microscopy

Images showed initial hydration with dispersed GA and lipid fragments, evolving into uniform, ~200 nm spherical liposomes with a bilayer envelope and entrapped drug.

Stability

After 6 months at 25 °C, EE remained 72.82 % and diameter 198 nm, indicating robust physical stability.

In‑Vitro Release

Both PBS and saline media exhibited sustained release: 65.25 % and 69.46 % of GA released in 12 h, with a rapid initial phase followed by a plateau, confirming sustained‑release behavior.

Cellular Uptake

HepG2 uptake increased from 1,480 ng mL⁻¹ protein at 30 min to 2,030 ng mL⁻¹ at 240 min, with fluorescence microscopy corroborating time‑dependent internalization.

Conclusions

This work demonstrates that the lyophilization monophase solution method, coupled with Box‑Benhnken design and trehalose lyoprotectant, produces GA‑loaded liposomes with high encapsulation efficiency (74.87 %), small particle size (191 nm), and sustained release over 12 h. The method yields a stable dry powder suitable for large‑scale production and offers a promising platform for delivering hydrophobic therapeutics.

Abbreviations

BBD

Box‑Benhnken design

DSC

Differential scanning calorimetry

EE

Encapsulation efficiency

FTIR

Fourier transform infrared spectroscopy

GA

Glycyrrhetinic acid

MD

Mean diameter

SPC

Soybean phosphatidylcholine

TBA

Tert‑butyl alcohol

TEM

Transmission electron microscopy

XRD

X‑ray diffraction