Optimized Preparation and Physicochemical Characterization of Dual‑Drug Nanoliposomes Encapsulating Erlotinib and Doxorubicin

Abstract

Synergistic combinatorial chemotherapy hinges on delivering drugs with distinct mechanisms in a coordinated fashion. We present a reproducible, non‑PEGylated nanoliposome platform that co‑encapsulates erlotinib (ERL) within the lipid bilayer and doxorubicin (DOX) in the aqueous core. Lipids (DSPC, cholesterol, POPG) were assembled by thin‑film hydration, followed by probe‑sonication to achieve sub‑200 nm vesicles. DOX was loaded using ammonium‑sulfate (AS) or pH gradients; the AS‑gradient yielded >90 % DOX encapsulation efficiency (EE) versus ~17 % for the pH method. ERL EE reached 30 %. Transmission electron microscopy revealed crystalline DOX‑sulfate cores and less‑ordered ERL crystals in the bilayer. The vesicles remained size‑stable across 4–37 °C and released ERL rapidly (<8 h) while DOX released slowly, enabling a time‑differential, synergistic profile. These findings establish a robust dual‑drug nanoliposome platform suitable for translational studies.

Background

Triple‑negative breast cancers and other refractory tumors resist standard monotherapies due to intertwined DNA‑damage and survival pathways. Combination regimens that first prime tumors with a growth‑factor inhibitor and then expose them to a genotoxic agent have shown preclinical synergy. To translate such in vitro effects clinically, a delivery vehicle that co‑delivers both agents and synchronizes their release is essential, especially when pharmacokinetics differ markedly.

Nanoliposomes—phospholipid bilayer vesicles—naturally encapsulate hydrophilic drugs in their core and hydrophobic drugs in their membrane, achieving co‑localization and enhanced tumor accumulation via the enhanced permeability and retention (EPR) effect. However, reliable, reproducible manufacturing protocols and comprehensive physicochemical data remain scarce, limiting clinical translation.

Methods

Materials

DSPC, POPG, cholesterol (Avanti Polar Lipids), DOX·HCl and ERL free base (Boryung Co. / Shanghai Send), ammonium sulfate, citric acid (Daejung), and reagents (Sigma‑Aldrich).

Preparation of Dual‑Drug Nanoliposomes

ERL was incorporated into a 27:20:3 (w/w/w) DSPC:cholesterol:POPG mixture (3:50 w/w drug) and dried to a thin film. Hydration with 250–400 mM AS (or 300 mM citric acid, pH 3.9) at 65 °C produced multilamellar vesicles. Probe sonication (5 s on/2 s off, 20 % amplitude, 36 J/pulse) in an ice‑water bath reduced the size to ~140 nm. Excess AS was removed by overnight dialysis against PBS (pH 7.4).

DOX (1.5 mg/mL, 2 mL) was added to 8 mL of the ERL‑loaded liposomes and loaded by either a pH‑gradient or an AS‑gradient. Four DOX‑loading protocols (incubation, sonication, equilibration, dialysis) were evaluated to optimize EE.

Characterization

Dynamic light scattering measured size and polydispersity; TEM (JEM‑2100F) visualized morphology and crystal structures. Encapsulation efficiency was calculated from drug released by Triton X‑100 and quantified by UV‑Vis (ERL) or fluorescence (DOX). In vitro release employed dialysis cassettes in PBS (pH 7.4) at 37 °C. Physical stability was assessed by monitoring size at 4, 25, and 37 °C over 21 days.

Results and Discussion

Size Control by Sonication

Untreated ERL liposomes were ~760 nm, shrinking to ~222 nm within 10 min of probe sonication; further sonication yielded negligible size changes. Water‑bath sonication achieved a similar reduction faster but increased heating, so ice‑water cooling was preferred. Post‑dialysis liposomes averaged 140 nm.

DOX Loading Efficiency

AS‑gradient loading produced 90 %+ EE, far exceeding the ~17 % from pH‑gradient. A 350 mM AS concentration maximized EE and minimized variability, likely due to favorable DOX‑sulfate crystallization. Among the four protocols, the combination of 65 °C incubation, 15 min probe sonication, and 30 min room‑temperature equilibration (Group 2) yielded the highest EE (~98 %). Longer sonication or delayed equilibration reduced EE, presumably by AS leakage.

Morphology and Crystallinity

TEM images confirmed spherical vesicles <200 nm. High‑resolution TEM and SAED revealed highly ordered DOX‑sulfate crystals in the core and less‑ordered ERL crystals at the bilayer outer surface. Single‑drug controls displayed similar crystalline patterns, validating the dual‑drug system’s structural integrity.

Physical Stability

Diameter changes were <15 % at 4 °C, <10 % at 25 °C, and <6 % at 37 °C over 21 days, indicating robust stability. DSPC’s high phase‑transition temperature and 40 % cholesterol content likely contributed to this resilience.

Time‑Differential Release

In PBS, ERL released ~65 % by 48 h, whereas DOX released ~30 %. The initial 8 h window showed >4 % h⁻¹ ERL release versus <1 % h⁻¹ DOX, confirming a sequential release profile conducive to synergistic action.

Conclusions

We established a scalable, non‑PEGylated nanoliposome platform that co‑encapsulates ERL and DOX with high EE and controlled size. Probe sonication after film hydration optimally reduces vesicle diameter. AS‑gradient loading, especially with 350 mM AS and a brief post‑sonication equilibration, yields the highest DOX EE. TEM and SAED confirm distinct crystalline arrangements of each drug. The vesicles are physically stable and exhibit a time‑differential release of ERL and DOX, positioning them as a promising vehicle for synergistic cancer therapy.

Abbreviations

DOX

Doxorubicin

DSPC

1,2‑Distearoyl‑sn‑glycero‑3‑phosphocholine

EE

Encapsulation efficiency

EPR

Enhanced permeability and retention

ERL

Erlotinib

PK

Pharmacokinetic

POPG

1‑Palmitoyl‑2‑oleoyl‑sn‑glycero‑3‑phospho‑(1′‑rac‑glycerol) (sodium salt)

SAED

Selected area electron diffraction

SEM

Standard error of mean

Tc

Phase‑transition temperature

TEM

Transmission electron microscopy