Anti-CD38 targeted nanotrojan horses stimulated by acoustic waves as therapeutic nanotools selectively against Burkitt's lymphoma cells

Abstract

The horizon of nanomedicine research is moving toward the design of therapeutic tools able to be completely safe per se, and simultaneously be capable of becoming toxic when externally activated by stimuli of different nature. Among all the stimuli, ultrasounds come to the fore as an innovative approach to produce cytotoxicity on demand in presence of NPs, without invasiveness, with high biosafety and low cost. In this context, zinc oxide nanoparticles (NPs) are among the most promising metal oxide materials for theranostic application due to their optical and semi-conductor properties, high surface reactivity, and their response to ultrasound irradiation. Here, ZnO nanocrystals constitute the stimuli-responsive core with a customized biomimicking lipidic shielding, resembling the composition of natural extracellular vesicles. This core-shell hybrid structure provides high bio- and hemocompatibility towards healthy cells and is here proofed for the treatment of Burkitt's Lymphoma. This is a very common haematological tumor, typically found in children, for which consolidated therapies are so far the combination of chemo-therapy drugs and targeted immunotherapy. In this work, the proposed safe-by-design antiCD38-targeted hybrid nanosystem exhibits an efficient selectivity toward cancerous cells, and an on-demand activation, leading to a significant killing efficacy due to the synergistic interaction between US and targeted hybrid NPs. Interestingly, this innovative treatment does not significantly affect healthy B lymphocytes nor a negative control cancer cell line, a CD38- acute myeloid leukemia, being thus highly specific and targeted. Different characterization and analyses confirmed indeed the effective formation of targeted hybrid ZnO NPs, their cellular internalization and the damages produced in Burkitt's Lymphoma cells only with respect to the other cell lines. The presented work holds promises for future clinical applications, as well as translation to other tumor types.

Keywords: Biomimetic nanoparticles; Monoclonal antibody fragments; Stimuli-responsive nanoparticles; Targeted therapy; Ultrasounds; Zinc oxide.

Fig. 1

Transmission Electron Microscopic images of a ZnO NCs and b ZnO-Lip. c DLS measurements and d Z-potential of ZnO and ZnO-Lip e Nanoparticle Tracking Analysis measurements of ZnO (black curve), ZnO-Lip (red curve) and ZnO-Lip (green curve) in water

Fig. 2

Cytotoxicity on Daudi cells at 24 h (a), and 48 h (b) of different concentrations of ZnO and ZnO-Lip. Cytotoxicity on Lymphocytes at 24 h (c), and 48 h (d) of different concentrations of ZnO and ZnO-Lip. Cytotoxicity on HL60 at 24 h (e), and 48 h (f) of different concentrations of ZnO and ZnO-Lip. All experiments were performed at least in duplicate. *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001

Fig. 3

a Cytotoxcity on Daudi, B-Lymphocytes (B-ly.) and HL60 cell lines treated with 40 ug/mL of ZnO-Lip or ZnO-LipCD38 after 24 h and 48 h. b ZnO-Lip or ZnO-LipCD38 internalized or bounded to Daudi, Lymphocytes and HL60 cell membranes 24 h and 48 h after the treatment. Liposomes were marked with DiD, and cells were treated with 40 μg/mL of nanoconstructs. All experiments were performed at least in duplicate. *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001

Fig. 4

3D fluorescence microscopy images of Daudi (a) and lymphocytes (d) control cells, Daudi treated with ZnO-Lip (b) and ZnO-LipCD38 (c) and lymphocytes treated with ZnO-Lip (e) and ZnO-LipCD38 (f) after 24 h. Liposome containing ZnO NCs were labelled with DiD (red channel); cell nuclei were labelled with Hoechst (blue channel); cell membranes were labelled with WGA488 (green channel). Cells were treated with 40 μg/mL

Fig. 5

Fluorescence microscopy images of the internalization and colocalization of ZnO-LipCD38 nanoconstruct on Daudi and lymphocytes after 24 h. Liposome containing ZnO NCs were labelled with DiD (red channel); antiCD38 fragments incorporated in the lipidic shell contained ZnO NCs were labelled with Curcumin (blue channel); cell membranes were labelled with WGA488 (green channel). Cells were treated with 40 μg/mL. White circles represent highly relevant internalization events in cells

Fig. 6

Cells viability of Daudi after 24 h (a) and 48 h (b) the treatment with US; Lymphocytes viability after 24 h (d) and 48 h (e) the treatment with US. US were produced by Lipo0 transducer, at 1 MHz, 100%DC. All the experiments were performed at least in triplicates. In US + ZnO-Lip and US + ZnO-LipCD38 groups, cells were previously incubated with 40 μg/mL of nanoconstructs 24 h before the US treatment. *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001

Fig. 7

Evaluation of the synergy derived from the combination of ultrasound irradiation and the administration of ZnO-Lip and ZnO-LipCD38 on Daudi, at 24 h (a) and 48 h (b), and on B-Lymphocytes, at 24 h (c) and 48 h (d). The effects generated by the administration of the two treatments (US and ZnO-Lip/ZnO-LipCD38) can be evaluated by the synergy score (δ); it can be < − 10 and it represents antagonistic interaction, > − 10 and < 10 and it represents additive interaction, or it can be > 10 and it represents synergistic interaction. The heatmaps highlight synergistic and antagonistic dose regions in red and green colors, respectively. Cells were previously incubated with 40 μg/mL of nanoconstructs 24 h before the US treatment The analysed data were at least in triplicate

Fig. 8

Evaluation of percentage of early and late apoptotic populations after 24 h (a), and 48 h (b) induced in Daudi cells after the US treatment with Lipo0 transducer (1 MHz, 100%DC, 0.45 W/cm², 1 min). In US + ZnO-Lip and US + ZnO-LipCD38 groups, cells were previously incubated with 40 μg/mL of nanoconstructs 24 h before the US treatment

Fig. 9

Evaluation of percentage of early and late apoptotic populations after 24 h (a), and 48 h (b) induced in B-Lymphocytes cells after the US treatment with Lipo0 transducer (1 MHz, 100%DC, 0.45 W/cm², 1 min). In US + ZnO-Lip and US + ZnO-LipCD38 groups, cells were previously incubated with 40 μg/mL of nanoconstructs 24 h before the US treatment

Fig. 10

Cells membrane integrity was evaluated with PI dye, while Zn²⁺ presence inside cell was measured by Fluozin dye. Percentage of cells positive to PI and Fluozin is shown for Daudi cells at 24 h (a), and 48 h (b) after treatment with US, and for B-Lymphocytes after 24 h (c), and 48 h (d) the US treatment. US were produced by Lipo0 transducer, at 1 MHz, 100%DC, 0.45W/cm², 1 min. 5 images per sample were considered, the two staining were evaluated separately. All the data are normalized with respect the total number of cells evaluated for each image (black column). In US + ZnO-Lip and US + ZnO-LipCD38 groups, cells were incubated with 40 μg/mL of nanoconstructs 24 h before the US treatment. *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001