Tromethamine‑Modified Chlorambucil Prodrug Nano-Micelles: Improved Colloidal Stability and Antitumor Efficacy

Chlorambucil (CLB) is a clinically effective alkylating agent for hematologic malignancies; however, its therapeutic application is severely hampered by poor aqueous solubility, rapid hydrolytic degradation in physiological fluids, and dose-dependent systemic toxicity. Conventional nanocarriers offer partial solutions but are limited by low drug loading (<10%) and complex fabrication. Prodrug strategy constructed by covalent modification of anticancer drugs can overcome these limitations, enabling improved physicochemical properties and tumor-specific drug release.

An amphiphilic prodrug (CLB-THAM) was synthesized by conjugating CLB to tromethamine (THAM), a pharmacopeial excipient with GRAS status. The prodrug was formulated into nanomicelles via nanoprecipitation and characterized by DLS and TEM. Hydrolytic stability, in vitro cytotoxicity against A20 lymphoma cells, apoptosis induction, cellular uptake, and in vivo antitumor efficacy in a syngeneic mouse model were systematically evaluated.

CLB-THAM spontaneously self-assembled into monodisperse nanomicelles (91.8 nm, PDI 0.103) with an unprecedented drug loading of 70.5%. Using A20 cells as experimental models, the cytotoxic effects of CLB-THAM nanomicelles were investigated at 0-100 μmol/L for 24 h. Notably, the cell viability of CLB-THAM nanomicelles to A20 cells was significantly lower compared with free CLB (45.6% vs. 74.5% at 100 μM), with correspondingly higher apoptosis induction (48.5% vs. 39.0% at 30 μg·mL^-1). In vivo, the nanoformulation achieved superior tumor suppression in A20 tumor-bearing mice (final tumor volume 421 mm³ vs. 610 mm³ for free CLB and 1424 mm³ for saline), while maintaining excellent hemocompatibility (hemolysis <5%) and minimal systemic toxicity as evidenced by body weight stability and histopathological analysis.

This carrier-minimized prodrug platform concurrently overcomes the solubility, stability, and toxicity bottlenecks of nitrogen mustard chemotherapy, offering a simple and translatable strategy for enhancing the clinical efficacy of chlorambucil.