Paclitaxel-loaded biodegradable ROS-sensitive nanoparticles for cancer therapy

Abstract

Background

Reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, trigger biodegradation of polymer-based nanoparticles (NPs) bearing pinacol-type boronic ester groups. These NPs may selectively release their cargo, in this case paclitaxel (PTX), at the high levels of ROS present in the intracellular environment of inflamed tissues and most tumors.

Purpose

The main objective was to determine anti-tumor efficacy of PTX-loaded ROS-sensitive NPs and to examine whether macrophage infiltration had any impact on treatment efficacy.

Methods

NPs were synthesized and their characteristics in the presence of H₂O₂ were demonstrated. Both confocal microscopy as well as flow cytometry approaches were used to determine degradation of ROS-sensitive NPs. HeLa cells were cultured in vitro and used to establish tumor xenografts in nude mice. In vivo experiments were performed to understand toxicity, biodistribution and anti-tumor efficacy of the NPs. Moreover, we performed immunohistochemistry on tumor sections to study infiltration of M1 and M2 subsets of macrophages.

Results

We demonstrated that PTX delivered in NPs containing a ROS-sensitive polymer exhibits a better anti-tumor efficacy than PTX in NPs containing ROS-non-sensitive polymer, free PTX or Abraxane^® (nab-PTX). The biodistribution revealed that ROS-sensitive NPs exhibit retention in liver, spleen and lungs, suggesting a potential to target cancer metastasizing to these organs. Finally, we demonstrated a correlation between infiltrated macrophage subsets and treatment efficacy, possibly contributing to the efficient anti-tumor effects.

Conclusion

Treatment with ROS-sensitive NPs containing PTX gave an improved therapeutic effect in HeLa xenografts than their counterpart, free PTX or nab-PTX. Our data revealed a correlation between macrophage infiltration and efficiency of the different antitumor treatments, as the most effective NPs resulted in the highest infiltration of the anti-tumorigenic M1 macrophages.

Keywords:

• reactive oxygen species
• ROS-sensitive nanoparticles
• paclitaxel
• treatment efficacy
• macrophage infiltration

Supplementary materials

Figure S1 Structure of polymers. Polymers synthesized and utilized for the preparation of the PTX-loaded P1 NPs (A) and P2 NPs (B). (C) AlexaFluor 647 conjugated NPs (only showed for P1)

Figure S2 Calculation of myeloid cell infiltration in tumor tissue. The displayed photomicrograph slide was digitalized and randomly selected frames were imported to ImageJ software. Subsequently, black threshold was created over original frame and a binary mask was created to visualize black spots on white background (A). Quantification of shown example displays stained pixelated area, total pixelated area, pixel size and pixel count. The ratio of the stained area pixel to the total area pixel is called the stained pixel ratio and is shown on the y-axes in Figures 6 and S6 (B)

Figure S3 Flow cytometry and confocal microscopy images in the presence of catalase. NR fluorescence ratio of released NR (red channel – FL5) vs entrapped NR (green channel – FL1) from P1 NPs (blue) and P2 NPs (green) without catalase or after the 16-hr pretreatment with catalase (A). Confocal images of NR-loaded P1 NPs (B) and P2 NPs (C) in PC-3 cells after a 16hr pretreatment with catalase. Red shows the fluorescence from released and quenched NR and green shows the fluorescence from the particle-trapped, non-quenched NR; the blue color shows the Hoechst-stained nucleus. Error bars show SD. Asterisks indicate statistical significance obtained by one-way ANOVA and Tukey’s multiple comparisons post-hoc test. *P<0.05, **P<0.01 (n=3)

Figure S4 Toxicity of P1 NPs, P2 NPs and nab-PTX in healthy mice. Weight change (%) in healthy mice after treating with 40 or 60 mg/kg PTX containing P1 NPs, P2 NPs or nab-PTX. Error bars show SEM

Figure S5 Photomicrographs of HeLa tissue samples labeled with CD68, iNOS or CD206. Photomicrographs of untreated tissue (A), and following treatment with P1 NPs (B), P2 NPs (C), nab-PTX (D) and PTX (E). Scale bar: 250 µm

Figure S6 Expression of the NK cell marker NCR1 quantified in P1 NPs and P2 NPs treated tumors. Calculation of the stained pixel ratio was performed as illustrated in Figure S2. Data are shown as mean-stained pixel ratio±SEM (n=5 random regions in each tumor). The difference was not statistically significant

Acknowledgments

We thank Markus Fusser for his help with the biodistribution experiments. We are grateful to the Department of Comparative Medicine for animal maintenance and support. The two groups at the Institute for Cancer Research in Oslo were supported by the Research Council of Norway under the program NANO2021, project number 228200/O70 (Biodegradable nanoparticles in cancer diagnosis and therapy). E Jäger, A Jäger, V Sincari and M Hrubý acknowledge the Czech Science Foundation (grant #17-09998S). P Štěpánek acknowledges financial support from Norwegian Grants (grant # 7F14009) and from the Ministry of Education, Youth and Sports (grant #LM2015064 ERIC; grant POLYMAT # LO1507).

Abbreviation list

AUC, area under the curve; DLS, dynamic light scattering; DMF, N,N-dimethylformamide; EPR, enhanced permeability and retention; FFPE, formalin-fixed and paraffin-embedded; Inos, inducible nitric oxide synthase; i.v., intravenously; nab-PTX, Abraxane^®; NK, natural killer; NPs, nanoparticles; PDI, polydispersity index; PTX, paclitaxel; SEM, standard error of the mean; TAMs, tumor-associated macrophages; TBS, tris-buffered saline.

Disclosure

Dr Martin Hrubý reports grants from Norwegian Grants, Ministry of Education, Youth and Sports of the Czech Republic, and Czech Science Foundation, during the conduct of the study. The authors report no other conflicts of interest in this work.