In vitro release profiles of MZ1-ACNPs at pH 5.8. conjugated with Trastuzumab by covalent coupling via zero-cross-linker carbodiimide chemistry to generate MZ1-loaded ACNPs (Scheme 1). Characterization of NPs and ACNPs was carried out by DLS and electronic microscopy (see Table 1, Physique 1, and Physique S3). DLS studies showed an average particle size for formulations close to 100 nm. The standard protocol of Bradford assay was employed for quantifying the concentration of the antibody in the supernatant (see Section 2). 1.6 nM was the trastuzumab cargo over the NP surface selected in accordance with previously published results . The trastuzumab conjugation was confirmed by the decrease in the surface charge of NPs (Z-potential) from +46.3 mV (MZ1-NPs) to +31.8 mV (MZ1-ACNPs). TEM images showed nanoparticles of the very comparable size reported by DLS steps and a core-shell morphology. After conjugation with trastuzumab, the surface of the NPs was altered (Physique 1). Open in a separate window Physique 1 TEM images of (A) MZ1-NPs and (B) MZ1-ACNPs. Table 1 Average size, polydispersity index (PdI), and Z-potential of the different formulation obtained by dynamic light scattering (DLS). 0.05; ** 0.01; *** 0.001. 3.3. Cell Cycle Arrest and Apoptosis Given the fact that MZ1-ACNPs inhibited cell proliferation in HER2+ breast malignancy cell lines, we next explored their mechanism of action. Thus, SKBR3 and BT474 were treated with vehicle, MZ1, MZ1-NPs, and MZ1-ACNPs for 48 h and 72 h for cell cycle and induction of apoptosis analysis, respectively. Concerning the cell cycle, the ACNPs did not show any statistically significant difference in relation to the distribution of the cell cycle phases (Physique 4A,B). On the other hand, the results concerning the induction of cell death, with a dose-treatment of L-Stepholidine MZ1-ACNPs of 50 nM, indicated a remarkable increase L-Stepholidine in the induction of apoptosis compared with the non-vectorized MZ1-NPs vehicle (Physique 4C,D). Open in a separate window Physique 4 MZ1-encapsulated nanoparticles do not affect cell cycle distribution in HER2+ cell lines and increase cell death by apoptosis induction in HER2+ cell lines. Distribution of cells (in % of the total) under treatment with vehicle, L-Stepholidine free MZ1, MZ1-NPs, or MZ1-ACNPs in SKBR3 TSPAN3 (A,C) and BT474 (B,D) HER2+ cell lines, evaluated by flow cytometry. ** 0.01; *** 0.001. 3.4. Cytotoxic Effect in HER2+, MZ1-Resistant Cell Lines Due to the strong cytotoxic effect observed with MZ1-ACNPs in the most MZ1-resistant cell line, its effect in a naturally MZ1-resistant HER2+ cell line, HCC1054, was assessed. MZ1-ACNPs were able to bypass natural resistance to this PROTAC, being the NPs more cytotoxic for this cell line after conjugation with trastuzumab (Physique 5). Open in a separate window Physique 5 MZ1-ACNPs rendered a strong cytotoxic effect in trastuzumab, MZ1-naturally resistant cell line HCC1954. Cell viability (in %, referred to the DMSO vehicle) by MTT assay under treatment with free MZ1, MZ1-NPs, or MZ1-ACNPs. *** 0.001. 4. Discussion Trastuzumab, pertuzumab, and T-DM1 are approved antibodies for the treatment of HER2+ breast cancer. Small molecule inhibitors of the kinase activity of the receptor, such as lapanitib or neratinib are also approved therapy for this disease. Both treatments improve clinical outcomes. However, there are still many patients who become resistant to treatment. Hence, HER2+ breast cancer remains cataloged as an incurable condition . Identification of novel and druggable targets remains a top priority for the pharma/biotech industry. PROTACs have great potential for L-Stepholidine therapeutic intervention , and their mechanism is based on the inhibition of protein function by hijacking a ubiquitin E3 ligase for protein degradation. However, the lack of tumoral.