Engineering Fluorescent Polyelectrolyte Microcapsules with Quantum Dot Encoding for Advanced Theranostic Applications

Abstract

Polyelectrolyte microcapsules are emerging as versatile carriers for drugs, imaging agents, and nanomaterials. Here we report a robust fabrication route that incorporates water‑soluble CdSe/ZnS quantum dots (QDs) stabilized with a tri‑functional polyethylene glycol (PEG) ligand into polyelectrolyte shells. The resulting QD‑encoded capsules were fully characterized by dynamic light scattering, electrophoretic mobility, scanning electron microscopy, and fluorescence/confocal microscopy. We quantified size distribution, surface charge, morphology, optical properties, and fluorescence lifetime dynamics, and we identified the optimal QD loading that maximizes brightness while preserving structural integrity. Finally, we demonstrate efficient internalization of the capsules by murine macrophages, confirming their suitability for live‑cell imaging and theranostic applications.

Background

Targeted delivery and imaging of therapeutics remain central to translational medicine. Polyelectrolyte microcapsules, assembled by layer‑by‑layer deposition of oppositely charged polymers, can encapsulate drugs, metal nanoparticles, or fluorescent probes, enabling controlled release and real‑time tracking of cargo [1–4]. By encoding these shells with semiconductor QDs, we obtain agents that simultaneously deliver therapeutic payloads and provide high‑contrast, photostable imaging signals [5–6].

The layer‑by‑layer (LbL) approach offers fine control over wall thickness, porosity, and surface chemistry, making it ideal for stimulus‑responsive systems [10–14]. QDs, with their broad absorption and narrow emission bands, allow multiplexed excitation from a single light source, outperforming conventional dyes in brightness and photostability [19–24].

Previous work has encoded polyelectrolyte capsules with organic dyes or carbon dots, but these strategies often require harsh thermal treatment or result in rigid, less responsive structures [25–30]. Our study introduces a gentler, aqueous QD transfer method that preserves capsule flexibility and responsiveness while delivering superior optical performance.

Experimental

Solubilization and Characterization of Quantum Dots

CdSe/ZnS core/shell QDs (λmax = 590 nm) were donated by Dr. Pavel Samokhvalov (MEPhI). The hydrophobic QDs were rendered water‑soluble by ligand exchange: TOPO was displaced with d,l‑cysteine, then replaced with HS–PEG12–COOH (Thermo Fisher). After successive washes, the QDs were dispersed in 0.1 M NaOH, sonicated, centrifuged, and filtered through a 0.22 µm membrane. QD concentration was determined spectrophotometrically at the first exciton peak, and the particles were stabilized by adding HS–PEG12–COOH at a molar ratio of 1:4.6, followed by 24–48 h incubation at 2–8 °C.

Synthesis of Calcium Carbonate Microparticles

Calcium carbonate microspheres were prepared by mixing equimolar Na2CO3 and CaCl2 solutions (0.33 M each) under controlled stirring (250, 500, 750 rpm). After 15–60 s, the mixture was quenched, washed with MilliQ water, and dried at 60 °C for 90 min. Optimal particle size (4–6 µm) and regular morphology were achieved at 250 rpm and 30 s stirring, as confirmed by SEM (Fig. 1).

Preparation of Polyelectrolyte Microcapsules Encoded with QDs

Calcium carbonate cores were first coated with five pairs of PAH/PSS layers (poly(allylamine hydrochloride) and poly(sodium 4‑styrenesulfonate)), terminating with a PAH layer. QDs were then adsorbed onto the positively charged surface, followed by additional PAH/PSS layers and a final PAH layer. The core was removed by three consecutive 15‑min incubations in 0.2 M EDTA (pH 6.5), yielding hollow microcapsules. For cell‑interaction studies, a final BSA coating (1 %) was applied to reduce non‑specific binding.

Characterization Techniques

Hydrodynamic diameter and ζ‑potential were measured by DLS and electrophoretic mobility (Malvern Zetasizer NanoZS). Fluorescence lifetimes were recorded using a pulsed YAG:Nd3+ laser (350 ps, 50 Hz) and a photomultiplier detector. QD loading was quantified by measuring unbound QDs in the supernatant. SEM (JEOL JSM‑7001F) visualized particle morphology, while fluorescence and confocal microscopy (Zeiss Axio Scope, Leica TCS SP5) assessed optical properties and cell uptake.

Cellular Uptake Studies

Murine alveolar macrophages (MH‑S) were incubated with ~1.2 × 10^6 BSA‑coated QD‑encoded capsules for 4 h (short‑term) or 24 h (long‑term). After counter‑staining nuclei with DRAQ5, confocal images revealed internalization of individual capsules and aggregates, with a higher uptake observed after prolonged exposure (Fig. 9).

Statistical Analysis

Data represent means ± SD from at least three independent experiments, analyzed with Excel and Origin Pro.

Results and Discussion

Synthesis and Characterization of Calcium Carbonate Microparticles

Optimal stirring conditions (250 rpm, 30 s) produced nearly spherical particles (4–6 µm) with minimal aggregation (Fig. 1). SEM images confirmed a porous surface composed of sub‑micron grains, suitable for subsequent LbL assembly.

Preparation and Characterization of QD‑Encoded Microcapsules

The QDs exhibited a narrow emission peak at 590 nm and a hydrodynamic diameter of ~25 nm (Fig. 2). Sequential PAH/PSS deposition inverted the surface charge as expected (Table 1). SEM images (Fig. 4) show progressive shell thickening, while fluorescence microscopy confirms the presence of QDs within the shell (Fig. 5). Confocal imaging (Fig. 6) reveals a clear hollow core, indicating successful core dissolution.

Encoding Efficiency

QDs were adsorbed onto the positively charged surface with a linear increase up to ~2.24 mg loading; higher concentrations led to saturation and reduced adsorption (Fig. 7a). Fluorescence intensity plateaued across the loaded range, providing sufficient contrast for imaging (Fig. 7b).

Fluorescence Lifetime

Pure QDs displayed the longest lifetime, which decreased upon incorporation into microparticles and further upon core removal (Table 2). Lifetimes stabilized after 48 h, suggesting a negligible long‑term photobleaching effect.

Cellular Interaction

Confocal images demonstrate efficient phagocytosis of single capsules and aggregates by MH‑S cells after both 4 h and 24 h incubation (Fig. 9). Capsules were clearly distinguishable from nuclei and often appeared attached to or inside cells, confirming their potential as live‑cell imaging agents.

Conclusions

We have established a scalable, aqueous method for incorporating water‑soluble, PEG‑stabilized QDs into polyelectrolyte microcapsules. The resulting capsules exhibit uniform size, robust fluorescence, and efficient cellular uptake, positioning them as promising theranostic platforms for targeted drug delivery and real‑time imaging.

Abbreviations

EDTA

Disodium ethylenediaminetetraacetate

PAA

Polyacrylic acid

PAH

Polycation poly(allylamine hydrochloride)

PEG

Polyethylene glycol

PSS

Polyanion poly(sodium 4‑styrenesulfonate)

QD

Quantum dot

RPMI medium

Roswell Park Memorial Institute medium

SEM

Scanning electron microscopy

TOPO

Trioctylphosphine oxide