High-Molecular-Weight Fractions of Spruce and Eucalyptus Lignin as a Perspective Nanoparticle-Based Platform for a Therapy Delivery in Liver Cancer

Abstract

The natural polymer, lignin, possesses unique biodegradable and biocompatible properties, making it highly attractive for the generation of nanoparticles for targeted cancer therapy. In this study, we investigated spruce and eucalyptus lignin nanoparticles (designated as S-and E-LNPs, respectively). Both LNP types were generated from high-molecular-weight (Mw) kraft lignin obtained as insoluble residues after a five-step solvent fractionation approach, which included ethyl acetate, ethanol, methanol, and acetone. The resulting S-and E-LNPs ranged in size from 16 to 60 nm with uniform spherical shape regardless of the type of lignin. The preparation of LNPs from an acetone-insoluble lignin fraction is attractive because of the use of high-Mw lignin that is otherwise not suitable for most polymeric applications, its potential scalability, and the consistent size of the LNPs, which was independent of increased lignin concentrations. Due to the potential of LNPs to serve as delivery platforms in liver cancer treatment, we tested, for the first time, the efficacy of newly generated E-LNPs and S-LNPs in two types of primary liver cancer, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), in vitro. Both S-LNPs and E-LNPs inhibited the proliferation of HCC cells in a dose-dependent manner and did not affect CCA cell line growth. The inhibitory effect toward HCC was more pronounced in the E-LNP-treated group and was comparable to the standard therapy, sorafenib. Also, E-LNPs induced late apoptosis and necroptosis while inhibiting the HCC cell line. This study demonstrated that an elevated number of carbohydrates on the surface of the LNPs, as shown by NMR, seem to play an important role in mediating the interaction between LNPs and eukaryotic cells. The latter effect was most pronounced in E-LNPs. The novel S- and E-LNPs generated in this work are promising materials for biomedicine with advantageous properties such as small particle size and tailored surface functionality, making them an attractive and potentially biodegradable delivery tool for combination therapy in liver cancer, which still has to be verified in vivo using HCC and CCA models.

Elucidating the mechanism behind and investigating the efficacy of Traditional Chinese Medicine and Traditional Tibetan Medicine in combination with standard therapeutics in hepatocellular carcinoma and cholangiocarcinoma in vitro

“…Crystal violet staining assay (CVSA) was performed as previously described ( Rudalska et al, 2014 ; Śliwka et al, 2016 ; Pylypchuk et al, 2022 ). Briefly, cells were first washed with 100 μl 1 × phosphate buffered saline (PBS) and fixed using 100 μl of 4% paraformaldehyde.…”

“…Cell counting kit-8 (CCK-8) assay (Sigma-Aldrich, United States) was used to determine cell proliferation and was performed using a manufacturer protocol, as recently described ( Pylypchuk et al, 2022 ). Cell proliferation was checked at two time points, 24 and 48 h post-incubation with TCM and TTM.…”

“…Cell proliferation was checked at two time points, 24 and 48 h post-incubation with TCM and TTM. 10 μl CCK-8 solution was added to each well, incubated for 2 hours, measured at an optical density (OD) 450 nm using an Infinite 200 PRO Nano Quant Tecan Microplate Reader (TECAN, CH-8708 Mannedorf, Switzerland) and analyzed using i-controlTM software, as described ( Zhou et al, 2018 ; Pylypchuk et al, 2022 ).…”

“…Senescence β-galactosidase assay (SA-β-Gal) was performed as previously described ( Kang et al, 2011 ; Cahu and Sola, 2013 ; Eggert et al, 2016 ; Pylypchuk et al, 2022 ). Briefly, cells were first washed with PBS (pH 7.2–7.4) and fixed with 2% formaldehyde and 0.2% glutaraldehyde solution in PBS (pH 7.2–7.4).…”

Stabilized lignin nanoparticles for templated assembly of silver nanoparticles and covalent functionalization in dispersion state

“…Considering the effect of temperature on hydroxyl content of hydroxymethylated lignin shown in this study, this trend is in line with the results of Pylypchuk et al who found that increasing the content of aliphatic hydroxyl groups of lignin can result in smaller particle sizes. [35,36] It is worth noting that as the particle size decreases the turbidity in the colloidal dispersion drops which is the result of smaller angles of light scattering (Figure 2c). SEM images confirm this trend.…”