Nanocomposite Sensor Rapidly Quantifies Liposomal Chemotherapy Encapsulation Efficiency Without Physical Separation

A research team from Guangxi Medical University Cancer Hospital and Zhejiang Sci-Tech University has developed an electrochemical sensing platform capable of directly measuring the encapsulation efficiency of liposomal daunorubicin without requiring prior physical separation of free and encapsulated drug molecules. In work published in the journal Frontiers in Chemistry, investigators Qian Jiang, Shuai Gu, Weizhong Tang, Fei Yan, and Qianying Chen addressed a persistent analytical challenge in nanomedicine manufacturing, where traditional workflows rely on multi-step separation techniques such as dialysis, ultracentrifugation, or centrifugal ultrafiltration to isolate unencapsulated therapeutic agents before performing quantification. By engineering a glassy carbon electrode modified with a vertically ordered mesoporous silica film grown on a conductive ternary carbon nanocomposite interlayer, the researchers demonstrated that unencapsulated anticancer molecules can be enriched and quantified electrochemically while intact liposomes and interfering macromolecules are physically excluded from the sensor surface. This analytical architecture preserves the delicate partitioning equilibrium between encapsulated and free drug compartments, bypassing sample manipulation steps that can distort analytical accuracy during pharmaceutical formulation analysis.

Liposomes serve as essential vesicular drug delivery systems composed of phospholipid bilayers, featuring internal aqueous compartments and hydrophobic membrane domains that accommodate therapeutic payloads of diverse physicochemical properties. Encapsulating therapeutic compounds within liposomes can protect active pharmaceutical ingredients from in vivo enzymatic degradation, enhance apparent aqueous solubility, extend systemic circulation lifetimes, alter physiological biodistribution, and mitigate non-specific exposure to healthy tissues, thereby improving therapeutic windows and curbing toxicity. Encapsulation efficiency, defined rigorously as the percentage of the initial or total drug payload retained inside the vesicle interior, represents a critical quality attribute directly governing formulation stability, dosing precision, pharmacological efficacy, and recipient safety. For daunorubicin, an anthracycline chemotherapeutic agent widely administered for hematological malignancies such as acute lymphoblastic leukemia and acute myeloid leukemia, rigorous quality control during formulation is particularly vital. Daunorubicin exerts potent antitumor effects by intercalating into double-stranded DNA structures, inhibiting topoisomerase II activity, and inducing intracellular reactive oxygen species, which collectively halt DNA replication and transcription; however, unencapsulated drug molecules exhibit limited tissue selectivity and cause severe dose-limiting cardiotoxicity and myelosuppression.

Conventional analytical protocols tasked with determining liposomal encapsulation efficiency follow a separate-then-quantify paradigm that presents notable operational vulnerabilities across pharmaceutical quality assurance laboratories. In current practice, free drug fractions are separated from intact liposomal carrier vesicles through dialysis, ultracentrifugation, centrifugal ultrafiltration, or size-exclusion gel filtration, followed by quantification using high-performance liquid chromatography-mass spectrometry, ultraviolet absorption spectroscopy, capillary electrophoresis, or fluorescence spectroscopy. Dialysis procedures necessitate lengthy equilibration periods lasting several hours, creating substantial operational latency while running the distinct risk of secondary drug leakage or dynamic redistribution across dialysis membranes. Analytical ultracentrifugation and centrifugal ultrafiltration expose delicate vesicles to intense mechanical shear forces that can deform or rupture bilayer membranes, while ultrafiltration membranes frequently suffer from non-specific surface adsorption and incomplete retention of target solutes. Although optical spectroscopy offers simpler operation than chromatography, optical measurements remain vulnerable to sample turbidity, light scattering from dispersed lipid vesicles, fluorophore self-quenching, and matrix absorption backgrounds. Crucially, the physical separation process itself can perturb the thermodynamic partitioning equilibrium between encapsulated and free drug fractions, skewing analytical results.

Electrochemical sensing presents an inherently attractive alternative to conventional chromatographic and optical techniques because the quinone moiety within daunorubicin undergoes reversible, proton-coupled redox transformations at electrode interfaces, producing measurable current signals. Furthermore, electrochemical instruments provide rapid analytical response profiles, negligible sample consumption, cost-effective instrumentation, and broad adaptability toward miniaturized, on-site quality evaluation. However, the direct electrochemical response of daunorubicin at unmodified conventional electrodes remains comparatively sluggish and weak, yielding ill-defined redox peaks that lack sufficient analytical sensitivity. Concurrently, real-world biological fluids and lipid dispersions contain abundant proteins, suspended lipids, cellular fragments, and surfactant species that induce rapid electrode fouling, severe non-specific surface adsorption, and substantial background current fluctuations that degrade measurement reliability. Developing a practical electrochemical sensor for direct formulation analysis therefore demands a multifunctional interfacial architecture that simultaneously accelerates electron transfer kinetics, concentrates the target analyte at the sensing interface, and repels complex macromolecular contaminants found within intact liposomal matrices.

To overcome these interfacial constraints, the investigators synthesized a complementary ternary carbon nanocomposite comprising two-dimensional graphene oxide, one-dimensional multiwalled carbon nanotubes, and zero-dimensional nitrogen-doped graphene quantum dots. Two-dimensional graphene oxide provides an extensive surface area and abundant oxygen-containing functional groups that facilitate stable dispersion in aqueous processing media, while subsequent electrochemical reduction partially restores the conjugated sp2 carbon network to enhance electrical conductivity while retaining residual oxygen functionalities for interfacial chemical coupling. However, isolated sheets of reduced graphene oxide tend to restack via strong interfacial pi-pi interactions, which diminishes accessible surface active area. The incorporation of one-dimensional multiwalled carbon nanotubes remedies this structural limitation by establishing physical bridges across adjacent graphene sheets, preventing aggregation and constructing continuous, three-dimensional conductive pathways throughout the composite. Concurrently, zero-dimensional nitrogen-doped graphene quantum dots populate the exposed surfaces and structural edges of both nanotubes and graphene sheets, introducing catalytic defect sites that accelerate interfacial redox reactions while the surrounding carbon framework prevents the ultrasmall quantum dots from detaching or clustering into inactive domains.

The sensor was fabricated by first drop-casting ten microliters of an aqueous dispersion containing the ternary carbon nanocomposite onto a mechanically polished glassy carbon electrode having a three-millimeter diameter, followed by infrared drying to form a stable, conductivity-enhancing adhesive foundation. Subsequently, vertically ordered mesoporous silica films were synthesized directly atop the carbon-modified working electrode using an electrochemically assisted self-assembly method. The precursor growth solution comprised cetyltrimethylammonium bromide surfactant templates and tetraethoxysilane dissolved in an ethanol and aqueous sodium nitrate mixture maintained at pH 2.6. Applying a constant cathodic polarization current density of negative 0.74 milliamperes per square centimeter for ten seconds drove silica polycondensation around self-assembled surfactant micelles while simultaneously inducing the in situ electrochemical reduction of underlying graphene oxide to electrochemically reduced graphene oxide. The resulting electrode was aged overnight at eighty degrees Celsius to crosslink the silica network, followed by chemical extraction of the surfactant micelles in an acidified ethanol solution for five minutes, leaving hexagonally packed, uniform nanochannels oriented perpendicular to the conductive carbon matrix with pore diameters measuring two to three nanometers and a film thickness of approximately ninety-six nanometers.

Transmission electron microscopy confirmed the structural integrity of the fabricated components, displaying semitransparent wrinkled graphene sheets interwoven with tubular carbon nanotubes and uniformly decorated with nitrogen-doped graphene quantum dots, alongside an intact, crack-free mesoporous silica film containing regular hexagonal pores. Electrochemical characterization using anionic ferricyanide and cationic ruthenium redox probes demonstrated that intact surfactant micelles completely passivated Faradaic electron transfer, confirming a pinhole-free film barrier across the composite interface. Following micellar extraction, the silica nanochannels displayed charge-selective permeability driven by the deprotonation of surface silanol groups above silica’s isoelectric point of approximately two, which imparted a net negative surface charge within the nanochannels at neutral and mildly acidic pH levels. This interfacial surface charge electrostatically repelled anionic ferricyanide probes while concentrating cationic ruthenium ions. Consequently, under mildly acidic measurement conditions where daunorubicin becomes protonated and positively charged, the mesoporous nanochannels selectively concentrated the therapeutic molecules into the active sensing zone while the narrow two-to-three-nanometer pore diameters mechanically excluded intact liposomes, which typically measure tens to hundreds of nanometers in diameter.

Comparative voltammetric evaluations revealed that while bare glassy carbon electrodes produced no discernible daunorubicin redox peaks within the tested potential window, and binary carbon variants produced only modest electrochemical currents, the integrated mesoporous film atop the ternary carbon composite generated sharp, well-defined redox responses. Driven by the synergistic combination of interfacial preconcentration within the silica nanochannels and accelerated electron transfer facilitated by the underlying carbon network, the differential pulse voltammetry oxidation peak current of daunorubicin at the optimized electrode demonstrated a forty-eight-fold enhancement relative to the unmodified glassy carbon substrate. Systematic experimental optimization established that composite precursor loadings of 0.10 milligrams per milliliter graphene oxide, 0.15 milligrams per milliliter multiwalled carbon nanotubes, and 0.10 milligrams per milliliter nitrogen-doped graphene quantum dots yielded peak catalytic amplification. Furthermore, investigating preconcentration times showed that signal currents increased progressively before plateauing at 420 seconds due to adsorption site saturation, while varying electrolyte pH revealed optimal signal intensity at pH 6.0, reflecting a mechanistic balance between drug protonation and silica nanochannel surface charge density.

Under optimized operating parameters using 0.1 molar phosphate-buffered saline at pH 6.0, differential pulse voltammetry measurements demonstrated two distinct linear detection intervals spanning 0.001 to 1.0 micromolar and 1.0 to 20.0 micromolar, with a calculated limit of detection of 514 picomolar based on a signal-to-noise ratio of three. Scan rate dependencies verified that the interfacial redox process was governed by surface-controlled kinetics involving equal numbers of protons and electrons, matching the classic two-electron, two-proton redox mechanism characteristic of anthracycline quinone structures. The sensor exhibited exceptional fabrication reproducibility across five independently prepared electrodes, displaying a relative standard deviation of 2.0 percent, alongside robust storage stability retaining 93.2 percent of initial response following seven days of refrigerated storage at four degrees Celsius. Selectivity assays demonstrated less than 3.2 percent current variation in the presence of challenging interferents, including sodium chloride, glucose, ascorbic acid, uric acid, dopamine, bovine serum albumin, and liposomal lipids. Furthermore, recovery tests conducted using spiked commercial human serum samples yielded analytical recoveries ranging from 96.6 to 99.2 percent with relative standard deviations between 0.5 and 1.1 percent, verifying matrix tolerance.

The practical utility of the platform was demonstrated by assessing distearoylphosphatidylcholine and cholesterol liposomes loaded with daunorubicin across a transmembrane pH gradient, where free drug content was quantified directly in intact liposomal suspensions diluted one-hundred-fold without preliminary filtration or centrifugation. The initial daunorubicin concentration of 1,000 micromolar yielded an unencapsulated drug reading of 82.0 micromolar, corresponding to an encapsulation efficiency of 91.8 percent, which showed remarkable agreement with a standard reference value of 91.4 percent obtained via conventional ultraviolet-visible spectrophotometry following physical extraction. Control experiments using blank liposomes mixed with daunorubicin in the absence of transmembrane loading confirmed negligible surface adsorption of 4 micromolar and verified that suspended liposomes did not interfere with voltammetric signals due to size exclusion by the silica nanochannel architecture. Furthermore, chemical disruption of dialyzed liposomes using a chloroform-methanol extraction mixture liberated 904 micromolar of daunorubicin, which closely matched the expected encapsulated fraction and confirmed formulation stability. By eliminating multi-step physical separations, this mesoporous electrochemical sensing platform establishes an efficient, accurate analytical strategy for quality control in liposomal drug manufacturing.

Subject of Research: Electrochemical determination of liposomal daunorubicin encapsulation efficiency using mesoporous silica films on ternary carbon nanocomposites

Article Title: Separation-free electrochemical determination of liposomal daunorubicin encapsulation efficiency using mesoporous film on ternary carbon nanocomposite

Article References: Jiang, Q., Gu, S., Tang, W., Yan, F., & Chen, Q. (2026). Separation-free electrochemical determination of liposomal daunorubicin encapsulation efficiency using mesoporous film on ternary carbon nanocomposite. Frontiers in Chemistry, 14, 1978992. https://doi.org/10.3389/fchem.2026.1978992

Image Credits: AI Generated

DOI: 10.3389/fchem.2026.1978992

Keywords: Electrochemistry, Biosensors, Daunorubicin, Liposomes, Nanocomposites, Mesoporous Silica, Graphene Oxide, Carbon Nanotubes, Separation-free, electrochemical, determination, liposomal