Glycogen-nucleic acid constructs for gene silencing in multicellular tumor spheroids

Abstract

The poor penetration of nanocarrier-siRNA constructs into tumor tissue is a major hurdle for the in vivo efficacy of siRNA therapeutics, where the ability of the constructs to permeate the 3D multicellular matrix is determined by their physicochemical properties. Herein, we optimized the use of soft glycogen nanoparticles for the engineering of glycogen-siRNA constructs that can efficiently penetrate multicellular tumor spheroids and exert a significant gene silencing effect. Glycogen nanoparticles from different bio-sources and with different structural features were investigated. We show that larger glycogen nanoparticles ranging from 50 to 80 nm are suboptimal systems for complexation of nucleic acids if fine control of the size of constructs is required. Our studies suggest that 20 nm glycogen nanoparticles are optimal for complexation and efficient delivery of siRNA. The chemical composition, surface charge, and size of glycogen-siRNA constructs were finely controlled to minimize interactions with serum proteins and allow penetration into 3D multicellular spheroids of human kidney epithelial cells and human prostate cancer cells. We introduced pH sensitive moieties within the construct to enhance early endosome escape and efficiently improve the silencing effect in vitro. Glycogen-siRNA constructs were found to mediate gene silencing in 3D multicellular spheroids causing ∼60% specific gene silencing. The optimized construct exhibited an in vivo circulation lifetime of 8 h in mice, with preferential accumulation in the liver. No accumulation in the kidney, lung, spleen, heart or brain, or signs of toxicity in mice were observed. Our results highlight the potential for screening siRNA nanocarriers in 3D cultured prostate tumor models, thereby improving the predictive therapeutic efficacy of glycogen-based platforms in human physiological conditions.

Glycogen as a Building Block for Advanced Biological Materials

“…For example, comparison of the H1–6 to the H1–4 proton signal and the H4 to the H1–4 signal of bovine liver glycogen reveals the particles have 9% branching and 12% chain ends defined as a percentage per monomer unit ( Table 1 ). It is worth noting that this approximation assumes that all protons in the glycogen nanoparticles can be probed by NMR spectroscopy. NMR studies of large molecules and polymers, such as glycogen, are typically complicated by an increased line width, which for glycogen is associated with a slower tumbling of chains in the inner core of the particle.…”

“…In contrast, commercial preparation methods likely use harsher conditions that may result in the more disperse β‐particulate glycogen. The ζ‐potential of most commercial glycogen particles is negative, depending on the source, spanning from neutral up to about −10 mV (Table ) . This charge is indicative of a small amount of residual phosphate from the biological synthesis/degradation routes.…”

“…Glycogen from these different sources have been also characterized in our laboratory in terms of physical properties such as particle size, degree of branching and chain ends, molecular weight, protein content, and degradability (Table ). Typically, the different biodegradability by α‐amylase (Table ), in terms of both extent and kinetics, correlates to the enzymatic penetrability of the particles. In vivo, the degradability is dictated by numerous other α‐glucosidase enzymes.…”

“…Mitani et al have shown that oral administration of ESG suppresses dextran sulfate sodium‐ and 2,4,6‐trinitrobenzenesulfonic acid‐induced colitis and oxidative stress in mice, suggesting that ESG could prevent inflammatory bowel disease. Interestingly, as natural glycogen is typically only present in the cytoplasm, there is limited data on the uptake pathway of exogenous glycogen nanoparticles into cells . A recent study by Ida‐Yonemochi et al showed that uptake of ESG into MC3T3‐E1 cells was accompanied with an increase in expression of the glucose transporter 1 (GLUT‐1) protein, and when this protein was inhibited, the ESG particles mainly aggregated on the cell surface, with some particles internalized by endocytosis (Figure G–I).…”

“…Chemical modifications can be preferentially performed in aqueous environments under mild conditions and using nontoxic components. The polysaccharide chains can be functionalized and the nanoparticles remain partially biodegradable . Together, these properties potentially make glycogen nanoparticles well suited for use as a functional nanomaterial in therapeutic applications.…”