Elsevier

Acta Biomaterialia

Volume 103, February 2020, Pages 213-222
Acta Biomaterialia

Full length article
siRNA release kinetics from polymeric nanoparticles correlate with RNAi efficiency and inflammation therapy via oral delivery

https://doi.org/10.1016/j.actbio.2019.12.005Get rights and content

Abstract

Despite many efforts in the rational design of nanoparticles (NPs) to overcome the biological barriers to small interfering RNA (siRNA) delivery for improving gene silencing efficiency, little is known about the correlations between siRNA release kinetics and RNA interference (RNAi) efficiency and inflammation therapy via oral delivery. On the basis of mannose-modified trimethyl chitosan-cysteine (MTC) polymers, seven types of MTC NPs containing tumor necrosis factor (TNF)-α siRNA were prepared through ionic gelation. The siRNA release kinetics from MTC NPs were finely tuned by adjusting the kinds and amounts of the crosslinkers involved. These MTC NPs exhibited no disparities in siRNA protection against enzymatic degradation in physiological fluids and cellular uptake in macrophages; however, they showed distinct in vitro siRNA release profiles and intracellular unpacking kinetics. MTC NPs with relatively rapid and sustained siRNA release were responsible for efficient, prompt, and prolonged RNAi, contributing to desired therapeutic efficacy in acute and chronic inflammatory murine models following oral delivery. However, MTC NPs insufficiently releasing siRNA could not elicit effective RNAi. Collectively, the present investigation might provide broad insights into the optimization of siRNA nanocarriers with respect to their release kinetics for improving RNAi efficacies aiming at different types of inflammatory diseases.

Statement of significance

siRNA release kinetics in the cytoplasm and pathological characteristics of diseases themselves determine the therapeutic efficacy of siRNA delivery. Herein, by adjusting the kinds and amounts of the crosslinkers involved, we developed seven types of MTC NPs containing TNF-α siRNA with distinct siRNA release kinetics. MTC NPs with relatively rapid and sustained siRNA release were responsible for prompt and prolonged RNAi, respectively, contributing to desired therapeutic efficacy in acute and chronic inflammation following oral delivery. These results might provide broad insights into the optimization of siRNA nanocarriers in respect to their release kinetics for improving therapeutic outcomes toward different clinical requirements.

Introduction

Small interfering RNA (siRNA) has emerged as a promising therapeutic strategy for the management of viral infections, cancer, and inflammation-associated diseases [1,2]. To obtain in vivo highly effective gene silencing efficacy, a large amount of smart nanocarriers responsive to pH, reactive oxygen species (ROS), and redox have been developed to overcome the extracellular and intracellular delivery barriers [3], [4], [5]. These nanocarriers could enhance the siRNA amount that is released in the cytoplasm of the target cell and effectively trigger subsequent mRNA recognition and RNA interference (RNAi) [6,7], leading to improved therapeutic outcomes. While the amount of siRNA released to its site of action plays an important role in the treatment of diseases, the duration of the RNAi effect determined by the siRNA release kinetics in the cytoplasm and pathological characteristics of diseases themselves might also determine the therapeutic efficacy of siRNA delivery [8,9]. Acute and chronic inflammatory diseases show significant differences in their pathological processes. In the acute period, neutrophils are present in the first few hours to days, while macrophages are recruited by further downstream signals to aid in repair. Failure to repair and recovery leads to chronic inflammation which lasts for a few days or longer [10]. Because all therapeutic compounds have their corresponding optimal concentration window in a specialized indication, it is highly desirable that the siRNA release kinetics is controlled in an on-demand fashion for balanced therapeutic and side effects [11]. Therefore, to achieve an optimal anti-inflammation therapy, we hypothesize that different siRNA release kinetics are needed to maintain the proper siRNA concentration within an efficient time frame in acute and chronic inflammation. However, to the best of our knowledge, although nanoparticles (NPs) releasing siRNA in a sustained manner were effectively applied for the treatment of one kind of disease, including chronic inflammation, cancer, and tissue damage, through mediating prolonged gene silencing [12], [13], [14], [15], few attempts have been made to clarify how siRNA release kinetics from polymeric NPs affect the treatment of different types of inflammatory diseases according to RNAi efficacy.

One strategy of adjusting the siRNA release kinetics is the design of siRNA carriers with various degradation profiles owing to the disparities in chemical structures [14,16], which is complex and time-consuming for the synthesis of a series of delivery carriers. The other one is the fine tuning of the compositions of delivery carriers including the addition of variable polyanions and the incorporation of various amounts of oil phase [12,17], which is relatively simple and easy to be manipulated. However, for these carriers, the cationic component involved should meet the requirement for reproducing high efficiency in mediating gene silencing, otherwise, their differences in the RNAi kinetics are difficult to be observed. We previously reported a NPs-based oral delivery vehicle for tumor necrosis factor (TNF)-α siRNA termed mannose-modified trimethyl chitosan-cysteine (MTC) NPs, which overcame various extracellular and intracellular barriers to oral RNAi to mediate effective TNF-α silencing in mice [18]. Therefore, we are inspired to exploit the platform of MTC-formulated NPs to understand the implications of siRNA release kinetics on the RNAi efficiency as well as the therapeutic efficacy in acute and chronic inflammation via the oral route.

In the present investigation, seven types of siRNA loaded MTC NPs were prepared by simple crosslinking of cationic MTC polymers with various amounts of tripolyphosphate (TPP), hyaluronic acid (HA), and methylacrylic acid-methyl methacrylate copolymer (Eudragit® S100, ES). The siRNA integrity in physiological fluids and cellular uptake of MTC NPs in Raw 264.7 cells (murine monocyte macrophages) was evaluated. Heparin sodium replacement, in vitro release, and intracellular unpacking kinetics demonstrated that the seven types of siRNA-loaded MTC NPs possessed distinct siRNA release kinetics. We correlated the siRNA release kinetics with the time-dependent in vitro gene silencing efficiency in Raw 264.7 cells up to 7 days. Further, we hypothesized that the siRNA release kinetics from MTC NPs was closely related to oral RNAi efficiency and anti-inflammation activity in the acute and chronic inflammatory diseases.

Section snippets

Materials, cell culture, and animals

MTC polymers with molecular weight (Mw) of 200 kDa, 11% mannose modification, 30% quarternization degree, free sulphydryl content of 109.7 ± 2.5 µmol/g, and disulfide content of 197.3 ± 0.6 µmol/g were prepared according to our previous report [18]. TPP, lipopolysacchride (LPS), d-galactosamine (D-GalN), ethidium bromide (EB), and heparin sodium were bought from Sigma (St. Louis, MO, USA). HA (Mw 100 kDa) and ES were provided by Zhenjiang Dong Yuan Biotech Co., Ltd. (Jiangsu, China) and Rohm

Results and discussion

Although siRNA has emerged as a promising strategy for disease treatment, the effects of siRNA release kinetics from the polymeric nanocarriers on the therapeutic outcomes of various diseases remain unexplored. We originally developed MTC NPs in the previous investigation and demonstrated that they took advantages of the three functional groups (trimethyl, thiol, and mannose) in promoting oral absorption and gene transfection to mediate effective TNF-α silencing in mouse oral administration [18]

Conclusions

By easily adjusting the kinds and amounts of crosslinkers, a series of crosslinker-involved MTC NPs with similar siRNA protection in physiological conditions and macrophage uptake showed various siRNA release profiles, which endowed the possibilities of gaining an understanding of the correlations between the siRNA release kinetics from polymeric NPs and RNAi efficiency and inflammation therapy mediated by orally administered NPs. The in vitro siRNA release of these MTC NPs was in accordance

Declaration of Interest Statement

The authors declare that they have no competing interest.

Acknowledgements

The authors are thankful for the financial support from the National Natural Science Foundation of China (Nos. 81072595, 81573356, and 81673371).

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