DEVELOPMENT OF NOVEL BIOTECHNOLOGICAL APPROACHES FOR THE DIAGNOSIS AND NANOTECHNOLOGY-BASED TREATMENT OF PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA: AN INTEGRATED MOLECULAR INVESTIGATION

Abstract

Pediatric acute lymphoblastic leukemia (ALL) is a biologically heterogeneous hematological malignancy characterized by the abnormal proliferation of immature lymphoid cells. Although improvements in molecular classification, measurable residual disease (MRD) monitoring, and chemotherapy have substantially improved outcomes, challenges remain in detecting molecular subtypes, monitoring treatment response, and reducing treatment-associated toxicity. Nanotechnology-based drug delivery systems may offer opportunities to improve the cellular delivery and release characteristics of anticancer agents. This study was designed to develop an integrated molecular diagnostic and nanotechnology-based therapeutic approach for pediatric B-cell acute lymphoblastic leukemia (B-ALL). The proposed strategy combined molecular detection of leukemia-associated genetic alterations with the formulation and in vitro evaluation of daunorubicin-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles. A molecular assay framework was established for detecting selected leukemia-associated fusion transcripts, including ETV6::RUNX1, BCR::ABL1, TCF3::PBX1, and KMT2A-rearranged transcripts. Daunorubicin-loaded PLGA nanoparticles were prepared using an oil-in-water emulsion-solvent evaporation method. Their physicochemical properties were evaluated using dynamic light scattering (DLS), zeta-potential analysis, transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FTIR). In vitro drug-release behavior, cellular uptake, cell viability, apoptosis, and expression of apoptosis-associated genes were included in the experimental framework. NALM-6 B-ALL cells were used as a leukemia cell model, while a non-malignant cell model was included to assess preliminary selectivity. In the illustrative dataset, daunorubicin-loaded PLGA nanoparticles had a mean hydrodynamic diameter of 186.4 ± 8.7 nm, a polydispersity index of 0.19 ± 0.03, and a zeta potential of −21.6 ± 2.4 mV. The simulated encapsulation efficiency was 78.6 ± 3.2%. The molecular assay framework showed illustrative sensitivities of 95.0–100% and specificities of 96.0–100%, depending on the target. Nanoparticle-associated daunorubicin produced concentration-dependent reductions in NALM-6 cell viability and increased Annexin V-positive cells. The illustrative gene-expression profile showed increased BAX and CASP3 expression and decreased BCL2 expression following treatment. Drug release was greater under the simulated acidic condition (pH 5.5) than at pH 7.4. These findings are hypothetical and require experimental validation. The proposed integrated strategy provides a framework for combining molecular characterization of pediatric B-ALL with polymeric nanoparticle-based drug delivery. Further investigation using independent patient samples, multiple leukemia cell lines, and appropriate in vivo models is necessary before diagnostic performance, therapeutic selectivity, or clinical benefit can be established. Acute lymphoblastic leukemia; pediatric cancer; molecular diagnosis; fusion genes; PLGA nanoparticles; daunorubicin; apoptosis; qRT-PCR; drug delivery; precision medicine.