Exploring the Anticancer Potential of Lamivudine-Loaded Polymeric Nanoparticles: In Vitro Cytotoxicity, Tissue Deposition, Biochemical Impact In Vivo, and Molecular Simulations Analysis

Abstract

Lamivudine is a synthetic nucleoside analogue to cytosine with a modified sugar moiety. It has potent action against Human Immunodeficiency Virus and chronic hepatitis. Recently, studies have also shown that lamivudine (3TC) can induce apoptosis in cancer cells and inhibit their proliferation, including breast cancer. We prepared polymeric nanoparticles using the double emulsification technique to incorporate polycaprolactone (PCL) as the polymer and lamivudine as the active compound. The nanoparticles were characterized by atomic force microscopy and dynamic light scattering. Then we carried out a full set of in vitro and in vivo analyses, including measurement of cytotoxicity, radiolabeling, biodistribution and biochemistry. The results showed the formation of 273 nm spherical nanoparticles with monodisperse behavior (PDI = 0.052). The radiolabeling with ^99mTc demonstrated the feasibility of the direct radiolabeling process. The cytotoxicity corroborated the potential against the triple-negative breast cancer line (MDA-MB-231). The biodistribution assay revealed high uptake in the liver, small and large intestines and bladder, besides the presence of nanoparticles in the urine. The in vivo biochemistry analysis showed alterations in some enzyme levels, including: alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma GT (GGT), creatinine (CRE), amylase (MAS), lactate dehydrogenase pyruvate (LDH-P) and glucose (GLU). Finally, we performed theoretical studies of molecular docking, molecular dynamics and interactions between lamivudine and key proteins regulating necroptosis, including epidermal growth factor receptor (EGFR), receptor-interacting protein kinase 1 (RIPK1), and receptor-interacting protein kinase 3 (RIPK3). Theoretical results showed lamivudine's adaptability to the binding sites of these proteins, with potential for optimization to enhance hydrophobic interactions and binding affinity. The findings demonstrated the efficacy of lamivudine against breast cancer cells, and the need to better understand the interplay of nanosystems with biochemical parameters.

Keywords: nanotechnology; oncology; radiolabeling; therapy; treatment.

1

Size by dynamic light scattering. The mean size of PCL nanoparticles for lamivudine nanoparticles. PdI: polydispersity index.

2

Atomic force microscopy of lamivudine films. A. 10-μm scan showing hundreds of lamivudine nanoparticlesthe blue dotted square denotes the region where the height map in Figure B was acquired; B. 1-μm scan showing details of nanoparticle surface; C. 3D map related to the image 2A; D. 3D map related to image 2B; E. Histogram of diameter distribution acquired in 10-μm maps. The mean diameter was 228.8 ± 1.7 nm (mean ± SD).

3

Release profile of lamivudine nanoencapsulated in polycaprolactone. (A) Pointwise (noncumulative) release curve showing a rapid increase in the first hours, followed by a stable release phase until the 30th hour, indicating a sustained release system. (B) Cumulative release profile, highlighting the progressive accumulation of the released mass over time. At the end of the experiment, the total release was 34.3 ± 0.4 mg of lamivudine, corresponding to 42.3% ± 0.5% of the initial mass. The data are expressed as mean ± standard deviation (SD).

4

Viability assay (MTT) of MDA-MB-231 lines treated with polycaprolactone (100 μg/mL) and lamivudine NPs at different concentrations (10, 50, and 100 μg/mL) for 24h PCL: polycaprolactone; LamNPs: lamivudine nanoparticles. Data are expressed as the percentage of the control group ± SD (***) p < 0.001, (**) p = 0.002 (LamNPs vs control); (###) p < 0.001, (##) p = 0.002 (LamNPs vs free lamivudine).

5

Biodistribution of ^99mTc-labeled lamivudine nanoparticles after 24 h, assessed in blood, urine, and various organs, including the heart, brain, stomach, large and small intestines, bladder, left and right kidneys, left and right lungs, liver, and spleen.

6

2D diagram of the interactions obtained for the best-ranked docking pose for interaction of crystallographic ligand AQ4 (A) and lamivudine (B) with EGFR; crystallographic ligand Q1A (C) and lamivudine (D) with RIPK1; crystallographic ligand ZOV (E) and lamivudine (F) with RIPK3. The main interactions of the ligands with the active site residues of the proteins are presented, highlighting the hydrogen bonds (dashed black lines) and the hydrophobic contact regions (green curved lines) to observe the critical binding sites involved in the stability of the complexes.

7

RMSD analysis of the complexes of EGFR, RIPK1 and RIPK3 with their respective ligands AQ4, Q1A, ZOV and lamivudine, as well as the apo forms of each protein, over MD simulation of 100 ns. The green curve represents the apo form, the blue curve denotes the complex with the specific ligand (AQ4 for EGFR, Q1A for RIPK1 and ZOV for RIPK3), and the red curve represents the complex with lamivudine. The RMSD variations over time reflect the conformational stability of the complexes, where larger fluctuations indicate greater flexibility and potential structural instability.