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Doxorubicin; Polymeric micelles; pH-responsive delivery; Poly(histidine); PEG; Tumor targeting; Drug release; Nanomedicine; Cancer therapy; Smart drug delivery

Introduction

Chemotherapeutic drugs such as doxorubicin (DOX) are effective but often associated with systemic toxicity due to non-specific biodistribution. Nanocarrier-based delivery systems offer the promise of tumor-selective accumulation via the enhanced permeability and retention (EPR) effect [1]. However, the EPR effect alone may not suffice to ensure controlled drug release at the tumor site. Incorporating environmental triggers—such as pH—into drug delivery systems can enhance tumor-specific release, as tumor extracellular pH tends to be mildly acidic (pH 6.5–6.8) compared to blood and healthy tissues [2].

Polymeric micelles formed by amphiphilic block copolymers are particularly suited for this purpose. Their core–shell architecture facilitates the solubilization of hydrophobic drugs while allowing surface functionalization [3]. Poly(histidine), a pH-sensitive polymer, undergoes conformational changes under acidic conditions due to the protonation of imidazole groups, leading to micelle destabilization and drug release [4]. In this study, we synthesize PEG-b-PHis copolymers and evaluate the resulting DOX-loaded micelles for their physicochemical properties, pH-responsive behavior, cellular uptake, and in vivo pharmacodynamics.

Materials and Methods

Methoxy-poly(ethylene glycol)-amine (mPEG-NHâ‚‚, 2 kDa), L-histidine-N-carboxyanhydride (His-NCA), and doxorubicin hydrochloride were obtained from commercial sources. PEG-b-PHis was synthesized by ring-opening polymerization of His-NCA initiated by mPEG-NHâ‚‚ in anhydrous DMF. The degree of polymerization was controlled to yield ~25 PHis units per PEG chain. DOX-loaded micelles were prepared by solvent evaporation, followed by dialysis against PBS (pH 7.4) to remove unencapsulated drug.

Micelle size and zeta potential were analyzed via dynamic light scattering (DLS). Transmission electron microscopy (TEM) was used to observe morphology. Drug loading and encapsulation efficiency were determined by UV–Vis spectroscopy (λ = 480 nm). pH-dependent drug release was studied in PBS at pH 7.4 and 6.5 over 48 hours. Cell viability was assessed using MTT assay on MCF-7 and HeLa cells at both pH values. Cellular uptake and intracellular distribution were examined using confocal laser scanning microscopy (CLSM). In vivo studies were performed in BALB/c nude mice bearing MCF-7 xenografts, and therapeutic efficacy was compared with free DOX (5 mg/kg, i.v., every 3 days for 15 days).

Results

The synthesized PEG-b-PHis copolymers formed stable micelles with a hydrodynamic diameter of 85.3 ± 4.6 nm and a polydispersity index of 0.18 ± 0.02. TEM revealed spherical morphology with uniform size. Zeta potential was -6.1 ± 0.9 mV, which contributed to colloidal stability and reduced nonspecific protein adsorption [5]. Encapsulation efficiency exceeded 92%, with a drug loading content of 10.6%.

In vitro drug release showed that less than 18% of DOX was released at pH 7.4 over 24 h, whereas at pH 6.5, 76% of DOX was released in the same time frame, confirming pH-triggered release behavior [6].

Cytotoxicity assays demonstrated that DOX-loaded micelles exhibited stronger inhibitory effects on cancer cells at pH 6.5 (ICâ‚…â‚€ = 0.87 µg/mL for MCF-7) compared to pH 7.4 (ICâ‚…â‚€ = 1.96 µg/mL), reflecting the pH-enhanced release. CLSM images showed rapid internalization of micelles and DOX accumulation in nuclei within 4 hours [7].

In vivo studies revealed significant tumor regression in the micelle-treated group compared to free DOX (p < 0.01). Mice treated with micelles showed no significant weight loss, and histopathological analysis indicated reduced cardiotoxicity, as confirmed by preserved myocardial architecture in H&E-stained heart tissues [8].

Discussion

The developed PEG-b-PHis micelles successfully harness tumor acidity to enable selective DOX release. The protonation of histidine residues at mildly acidic pH leads to destabilization of the micelle core, enhancing drug diffusion at the tumor site. This pH-triggered mechanism improves site-specific delivery, minimizing premature release in systemic circulation [9].

Compared to conventional PEG-PLA micelles or liposomes, PEG-b-PHis offers superior responsiveness and higher encapsulation efficiency. The micelle size (~85 nm) is well-suited for EPR-based tumor accumulation while being small enough to evade rapid clearance. Minimal surface charge helps in prolonging circulation time and reducing immunogenicity [10].

The micelles demonstrated enhanced therapeutic efficacy and safety in vivo. Their performance suggests great promise for clinical translation, particularly for solid tumors characterized by acidic extracellular pH. The synthetic route is scalable, and materials used are biocompatible and potentially FDA-approvable.

Conclusion

pH-responsive PEG-b-PHis micelles represent a robust and efficient nanocarrier for targeted delivery of doxorubicin. Their stability in circulation, selective release under tumor-like pH, and favorable safety profile offer significant advantages over conventional formulations. This platform may be extended to other pH-sensitive or hydrophobic anticancer agents for improved therapeutic outcomes.

Conflicts of Interest

The author declares no conflicts of interest.