Redox‑Responsive Dual‑Targeting Nanoparticles Co‑Deliver Curcumin to CD44‑Positive Tumor Mitochondria

Abstract

We engineered a multifunctional micellar system that simultaneously targets CD44 receptors on tumor cells and the mitochondria within those cells. The carrier, designated TPP‑oHA‑S‑S‑Cur, is assembled from (5‑carboxypentyl)triphenylphosphonium bromide (TPP), oligomeric hyaluronic acid (oHA), a redox‑cleavable disulfide linker, and curcumin (Cur). TPP confers mitochondrial accumulation, oHA delivers CD44‑mediated endocytosis, and the disulfide bond provides glutathione (GSH)‑responsive release. Characterization by ^1H‑NMR confirmed the successful synthesis of each component. Self‑assembly in aqueous media produced micelles with a mean diameter of 122.4 ± 23.4 nm and a zeta potential of −26.55 ± 4.99 mV. In vitro studies revealed pronounced redox sensitivity and dual‑targeting capability, highlighting this platform as a promising strategy to improve curcumin’s solubility, reduce systemic toxicity, and enhance therapeutic potency against cancer.

Background

Conventional chemotherapeutics often suffer from poor solubility, rapid clearance, and off‑target toxicity. Nanocarrier systems that can increase drug bioavailability, enable active targeting, and provide stimulus‑responsive release have therefore attracted intense research interest. Curcumin, a naturally occurring diphenolic compound isolated from Curcuma longa, displays broad anticancer activity but is limited clinically by its hydrophobicity, chemical instability, and low oral bioavailability. Previous efforts to encapsulate curcumin in polymeric micelles or lipidic nanoparticles have improved its aqueous solubility and extended circulation time; however, most formulations lack tumor‑specific delivery or intracellular release control.

Oligomeric hyaluronic acid (oHA) is a short HA fragment that retains excellent hydrophilicity, biocompatibility, and the ability to bind the CD44 receptor, which is overexpressed on many solid tumors, including breast and lung cancers. CD44‑mediated endocytosis offers a route for selective uptake by malignant cells. Moreover, mitochondria are critical regulators of apoptosis, and mitochondrial targeting has been shown to potentiate anticancer efficacy. Lipophilic cations such as triphenylphosphonium (TPP) accumulate selectively in mitochondria due to the organelle’s negative membrane potential.

Intracellular concentrations of glutathione (GSH) are typically 2–10 mM in tumor cells, markedly higher than in normal tissues. Disulfide bonds are cleaved efficiently by GSH, providing a mechanism for controlled drug release within the tumor microenvironment. Combining these concepts, we designed TPP‑oHA‑S‑S‑Cur as a dual‑targeting, redox‑responsive nanocarrier capable of delivering curcumin to CD44‑positive tumor cells and their mitochondria while enabling rapid drug release in response to elevated GSH levels.

Materials and Methods

Materials

Curcumin (Shanghai Zhanyun Chemical Co. Ltd), oHA (Mn = 10 kDa, Shandong Freda Biological Engineering Co. Ltd), 3,3′‑dithiodipropionic acid (Adamas Reagent Co. Ltd), TPP bromide, THF, DMSO, TEA, EDC, DMAP, and analytical‑grade reagents were obtained from standard suppliers.

Synthesis of TPP‑oHA‑S‑S‑Cur

The synthesis proceeded in three steps (Figure 2). First, TPP was esterified with oHA via EDC/DMAP coupling in DMSO to produce TPP‑oHA. Second, 3,3′‑dithiodipropionic acid was activated with oxalyl chloride, then coupled with curcumin under TEA catalysis to form S‑S‑Cur. Finally, S‑S‑Cur was conjugated to TPP‑oHA through another EDC/DMAP‑mediated reaction, yielding the final amphiphilic polymer. Dialysis (MWCO 2000 Da) and lyophilization removed unreacted small molecules. ^1H‑NMR spectra (Figure 3) confirmed the appearance of characteristic TPP aromatic peaks (7.57–7.72 ppm) and the S‑S‑Cur methylene signal at 2.50 ppm, establishing successful synthesis.

Micelle Preparation and Characterization

TPP‑oHA‑S‑S‑Cur (10 mg) and curcumin (1.5 mg) were dissolved in formamide (6 mL), then dialyzed against deionized water (MWCO 2000 Da) for 24 h in the dark to remove solvent. Centrifugation at 2500 rpm for 10 min removed any unencapsulated curcumin. The resulting suspension was filtered (0.45 µm) to yield Cur/TPP‑oHA‑S‑S‑Cur micelles. Size, polydispersity index (PDI), and zeta potential were measured by dynamic light scattering (DLS) on a Delsa Nano C system; morphology was examined by TEM (Hitachi H‑600). Drug loading (DL) and entrapment efficiency (EE) were quantified by HPLC at 425 nm following filtration (0.45 µm).

Redox‑Stimulated Release

Release profiles were evaluated by dialysis against PBS (pH 7.4, 45 % FBS, 0.5 % Tween 80) containing 0, 2, or 10 mM GSH at 37 °C. Samples were collected at defined time points up to 120 h and analyzed by HPLC. Curcumin release increased markedly with GSH concentration, confirming disulfide bond cleavage.

Cellular Studies

MDA‑MB‑231 breast cancer cells (high CD44 expression) were cultured in DMEM with 10 % FBS. Cytotoxicity was assessed by MTT after 24 h exposure to free curcumin, Cur/oHA‑Cur micelles, or Cur/TPP‑oHA‑S‑S‑Cur micelles at 0.5–40 µg/mL. Cellular uptake and mitochondrial localization were visualized by confocal fluorescence microscopy, using the intrinsic fluorescence of curcumin and MitoTracker Red CMXROS staining. Flow cytometry quantified mean fluorescence intensity (MFI) of cells incubated with each formulation over time.

Results and Discussion

Carrier Characterization

Figure 2 outlines the synthetic route; ^1H‑NMR spectra (Figure 3) confirm each conjugation step. Micelles displayed uniform, spherical morphology with a mean diameter of 122.4 ± 4.6 nm and a low PDI of 0.132 (Figure 4). The zeta potential of −21.56 ± 1.46 mV for Cur/TPP‑oHA‑S‑S‑Cur micelles indicates enhanced colloidal stability compared with Cur/oHA‑Cur micelles (−19.17 ± 0.55 mV). Drug loading and EE were 7.8 % and 94.3 %, respectively, surpassing conventional micelles.

Redox‑Triggered Release

In PBS lacking GSH, only 32.5 % of curcumin was released over 120 h, whereas the presence of 2 mM or 10 mM GSH accelerated release to 57.5 % and 75.3 %, respectively (Figure 5). This GSH‑dependent release demonstrates the system’s ability to liberate drug preferentially within the reductive tumor environment.

Cytotoxicity

MTT assays (Figure 6) revealed that Cur/TPP‑oHA‑S‑S‑Cur micelles exhibited the lowest IC_50 (3.87 µg/mL) compared with free curcumin (6.53 µg/mL) and Cur/oHA‑Cur micelles (5.09 µg/mL), indicating enhanced intracellular potency likely due to combined CD44 and mitochondrial targeting.

Cellular Uptake and Mitochondrial Localization

Fluorescence microscopy (Figure 7) showed rapid, time‑dependent accumulation of Cur/TPP‑oHA‑S‑S‑Cur micelles in MDA‑MB‑231 cells, with higher intensity than Cur/oHA‑Cur micelles. Pre‑incubation with free HA (2 mg/mL) reduced uptake, confirming CD44‑mediated entry. Co‑staining with MitoTracker Red revealed strong colocalization of curcumin fluorescence with mitochondrial markers (Figure 8), validating mitochondrial targeting via TPP.

Flow Cytometry

MFI measurements (Figure 9) mirrored microscopy observations, with Cur/TPP‑oHA‑S‑S‑Cur micelles achieving significantly higher fluorescence at all time points (p < 0.05).

Conclusions

We have developed a redox‑responsive, dual‑targeting nanoparticle platform that delivers curcumin to CD44‑positive tumor cells and their mitochondria, enabling controlled release in the presence of high GSH. The TPP‑oHA‑S‑S‑Cur micelles exhibit superior drug loading, stability, and cellular uptake, translating into enhanced cytotoxicity in vitro. This platform addresses curcumin’s solubility and bioavailability limitations and provides a versatile vehicle for hydrophobic anticancer agents. Future in‑vivo studies will assess therapeutic efficacy and biodistribution.

Abbreviations

oHA

Oligomeric hyaluronic acid

S‑S‑Cur

Dithiodipropionic acid‑curcumin conjugate

TPP

(5‑Carboxypentyl)triphenylphosphonium bromide