Acidic Micro‑Environments in Liposomes Significantly Enhance Curcumin Stability and Anticancer Efficacy

Background

Artificial lipid vesicles, or liposomes, emulate cellular membranes with a phospholipid bilayer surrounding an aqueous interior, offering high drug loading, biodegradability, and biocompatibility ^[1‑5]. Their amphiphilic architecture allows solubilization of hydrophobic drugs while shielding them from the harsh extracellular milieu ^[6‑9]. Surface modification further prolongs systemic circulation or directs payloads to specific tissues ^[10‑14], and several liposomal formulations have achieved clinical approval ^[15‑18].

Despite these advantages, delivering drugs that are unstable at neutral pH remains problematic. Conventional liposomes are typically prepared in phosphate‑buffered saline (PBS) at pH 7.4, thereby exposing pH‑sensitive cargos to an unfavorable environment. Curcumin, a lipophilic polyphenol with potent anti‑inflammatory and anticancer properties, exemplifies this issue: its poor aqueous solubility and pH‑mediated degradation limit clinical translation ^[19‑23]. Therefore, creating an acidic micro‑environment within the liposomal core could preserve the integrity of such drugs.

In this study, we leveraged the inner aqueous compartment of liposomes to generate a tunable acidity gradient and assessed its impact on curcumin encapsulation, stability, and anticancer activity.

Methods

Materials

Phosphatidylcholine from soybean lecithin (injection grade) was sourced from Shanghai Tai‑Wei Pharmaceutical Co., Ltd.; cholesterol was purchased from Amresco; poloxamer 188 (F68) was donated by BASF (China); curcumin (≥ 98 %) was supplied by Sigma; fetal bovine serum (FBS) came from HyClone. All reagents were of analytical grade.

Preparation of Curcumin‑Loaded Liposomes

Cur‑LPs were produced via the thin‑film evaporation method ^[27,28] with modifications. Phospholipids (75 mg) and cholesterol (5 mg) were dissolved in 0.5 mL ethanol containing 2 mg/mL curcumin. The solution was mixed with 5 mL 0.001 M PBS (pH 2.5, 5.0, or 7.4) containing 1 % (w/v) F68 to narrow size distribution. After 1 min magnetic stirring at 35 °C, the mixture was evaporated under vacuum and in darkness for 30 min to remove ethanol. Subsequent low‑speed centrifugation (3,000 rpm, 5 min) removed free curcumin; the pellet was then high‑speed centrifuged (16,000 rpm, 10 min) and resuspended in PBS (pH 7.4). The resulting formulations were designated Cur‑LP‑2.5, Cur‑LP‑5.0, and Cur‑LP‑7.4; blank liposomes were prepared identically.

Characterization

Dynamic light scattering (DLS) measured hydrodynamic size and polydispersity index (PDI); zeta potential was assessed by electrophoretic light scattering (ELS) using a Malvern Zetasizer Nano ZS90 at 25 °C. Encapsulation efficiency (EE) was calculated via fluorescence after separating encapsulated from free curcumin by high‑speed centrifugation ^[29].

Scanning Electron Microscopy

SEM images (FEI INSPECT F) were obtained after 100‑fold dilution in distilled water, drop‑casting on glass, air‑drying, and gold coating.

Physical Stability

Colloidal stability was evaluated by monitoring size changes and transmittance (550 nm) over 72 h at 37 °C. Aggregation (thermodynamic) and sedimentation (kinetic) were quantified respectively.

In Vitro Release

Curcumin release from Cur‑LPs was measured using a 10 kDa dialysis bag in PBS (pH 7.4, 0.1 % Tween 80) at 37 °C with 100 rpm stirring. Samples were collected at predetermined intervals, replaced with fresh medium, and analyzed by fluorescence (Ex 458 nm, Em 548 nm).

Chemical Stability in Serum

Cur‑LPs (10‑fold diluted) were mixed with 1 mL 50 % FBS and incubated at 37 °C, 100 rpm. At set times, aliquots were extracted, mixed with ethanol, centrifuged, and the remaining curcumin quantified by fluorescence.

In Vitro Anticancer Efficacy

HepG2 cells were seeded at 10,000 cells/well in 96‑well plates, cultured 24 h in PRIM‑1640 + 10 % FBS, washed with PBS, and then treated with 4 µg/mL curcumin (free or encapsulated) in serum‑free medium. Cell viability was assessed after 1 and 3 days using the CCK‑8 assay. Viability was expressed as % relative to untreated control.

Statistics

Data are presented as mean ± SD (n = 3). Two‑tailed Student’s t‑test determined significance; p < 0.05 was considered significant.

Results and Discussion

Characterization of Liposomes

All Cur‑LPs exhibited similar particle sizes (~300 nm) and PDI < 0.2 (Figure 1a). Zeta potentials were more negative for Cur‑LP‑5.0 and Cur‑LP‑2.5 (~ −18 mV) compared to Cur‑LP‑7.4 (−9 mV), reflecting the influence of internal acidity on surface charge (Figure 1b). The higher EE observed for Cur‑LP‑2.5 (74 %) versus Cur‑LP‑5.0 (45 %) and Cur‑LP‑7.4 (64 %) suggests that acidified interiors enhance curcumin loading, possibly due to altered solubility dynamics at low pH ^[33].

SEM imaging confirmed spherical morphology across all formulations (Figure 2). Minor aggregation was noted for Cur‑LP‑7.4, consistent with its lower zeta potential, and the observed SEM diameters were smaller than DLS values due to dehydration effects.

Physical Stability

Over 72 h at 37 °C, none of the formulations showed significant size increase or transmittance change (< 10 %), indicating robust thermodynamic and kinetic stability irrespective of internal pH (Figure 3).

In Vitro Release

Curcumin released rapidly from free solution (> 80 % within 6 h), confirming the dialysis membrane did not impede diffusion. Cur‑LPs displayed sustained, nearly zero‑order release, with ~30 % cumulative release over 72 h (Figure 4). Linear regression (R² > 0.99) underscored the consistent release kinetics, an advantageous feature for maintaining therapeutic levels.

Effect of Internal pH on Chemical Stability

Serum incubation revealed a pronounced pH dependence: after 6 h, Cur‑LP‑2.5 retained 55 % of curcumin, compared to 43 % (Cur‑LP‑5.0) and 34 % (Cur‑LP‑7.4) (Figure 5). This trend mirrors the known pH sensitivity of free curcumin ^[26]. The acidic core likely suppresses enzymatic activity and protects curcumin within the lipid bilayer, which, contrary to expectation, is permeated by a small aqueous volume that can influence drug stability.

In Vitro Anticancer Efficacy

Blank liposomes modestly promoted HepG2 proliferation at 1 day, suggesting a nutritional effect ^[27]. Free curcumin exhibited limited cytotoxicity due to poor solubility. In contrast, Cur‑LPs significantly inhibited cell growth in a pH‑dependent manner: after 1 day, viability was 80 % (Cur‑LP‑2.5 & Cur‑LP‑5.0) versus 90 % (Cur‑LP‑7.4); after 3 days, viability dropped to 24 % (Cur‑LP‑2.5), 21 % (Cur‑LP‑5.0), and 39 % (Cur‑LP‑7.4) (Figure 6). The superior performance of Cur‑LP‑2.5 aligns with its higher EE and stability.

Conclusions

Modulating the internal acidity of liposomes offers a straightforward yet powerful strategy to enhance the chemical stability and therapeutic efficacy of pH‑sensitive drugs, even for hydrophobic molecules that reside in the lipid bilayer. The acidic micro‑environment created in Cur‑LP‑2.5 not only preserved curcumin against serum degradation but also amplified its anticancer activity in HepG2 cells. This approach can be readily extended to other labile therapeutics, potentially improving their clinical performance.