Biophysical properties of chitosan/siRNA polyplexes: Profiling the polymer/siRNA interactions and bioactivity
Graphical abstract
Novel bioactive chitosan/siRNA nanoparticles were devised using Agaricus bisporus derived chitosans at low N:P ratios (4–8) characterized by low toxicity and good bioactivity in vitro.
Introduction
For decades biologists attributed a messenger role to RNA in the protein production machinery of the cell. This view changed with the discovery of Altman and Cech that RNAs can also be enzymatically active [1], [2]. Two and a half decades later Fire and Mello demonstrated that RNA can also function as a regulator of gene expression. This discovery sparked increased attention to this class of nucleic acid and resulted in the terminology RNA interference (RNAi)—the process of target sequence specific gene silencing mediated by catalytically active short-interfering RNAs (siRNA) [3]. Both groundbreaking discoveries were awarded with the Nobel price in 1989 and 2006 respectively.
To date delivering siRNA to the biologically relevant cellular organelle remains a key challenge. The complexity of this task can only be solved if the delivery vehicles give fully reproducible results and their physicochemical properties are thoroughly understood. Among the many nonviral delivery vehicles described in the literature are polymer based systems, which are mostly cationic in nature [4]. The underlying principle, which is complexing the siRNA via electrostatic interactions using an excess of the positively charged species (like the polymer) is well established. In that way mostly sub-micron particles are generated. In this work we selected chitosans as cationic polymers to interact with siRNA to form nanoparticles, referred to as polyplexes. Chitins are a structural element in the exoskeleton of crustacea (crabs, shrimp, etc.) and cell walls of fungi. Treated with HCl, chitosans are generated by a deacetylation process. Chitosans have found use as delivery vehicles for nucleic acids [5]. However previous work on chitosan/siRNA mostly used crustaceae derived polymer [6], [7], [8], [9], [10], [11], [12], [13] and mainly applied high N:P ratios (50–310), whereas only few studies investigated fungi derived chitosans [14], [15].
Here we used chemically unmodified chitosans extracted from the cell wall of fungus Agaricus bisporus [16], and describe the formulation of nanoparticles at low N:P ratios (~ 4–8). To understand the correlation between physicochemical properties of the polyplex and biological efficacy, four different chitosans of different molecular weight were characterized by quantitative gel permeation chromatography GPC. The effect of average molecular weight, polydispersity, hydrodynamic radius, intrinsic viscosity and thermodynamic polymer/siRNA interactions was investigated and related to in vitro potency by using the constitutively expressed luc reporter gene in human epithelial H1299 cells. We also investigated the intracellular trafficking of the two biologically most different polyplexes to further elucidate the impact of polyplex formulation parameters on biological performance. Structural features of the two most distinct polyplexes were mapped out using transmission electron microscopy (TEM), cryo-TEM and small angle X-ray scattering (SAXS) and relations to the bioactivity were made.
Section snippets
siRNAs and chitosans
Unmodified siRNA luc Gl3 (5 nM) was purchased from Qiagen (Germany); MW 14'8256 Da, sense: 5′-CUU ACG CUG AGU ACU UCG AdTdT-3′; antisense: 5′-UCG AAG UAC UCA GCG UAA GdT dT-3′. For confocal microscopy analysis a rhodamine labeled luciferase siRNA was purchased from Qiagen (Germany); MW 16204 Da; sense; 5′-AUGGAACCGCUGGAGAGCAACdTdT-rhodamine 3′; antisense: 5′-UGCUCUCCAGCGGUUCCAUCUdTdT-3′. As a negative control siRNA a published FVII sequence that is not expressed in H1299 cells was used [17].
Evaluation of polymer physical properties
Four chitosan batches were characterized with respect to molecular weight, intrinsic viscosity and hydrodynamic radius. Table 1 summarizes the physical properties of the polymer batches, which differed significantly in their average absolute molecular weights of 44, 63, 93 and 143 kDa respectively. The determined molecular weights are the absolute molecular weights due to the experimental set up. The molecular weight distribution increased slightly with increasing polymer size and so did the
Discussion
We focused on chemically unmodified, fungal chitosans with similar deacetylation degrees spanning a molecular weight range from 44 to 143 kDa. Formulations of siRNA polyplexes at low N:P ratios were devised and the polyplex physicochemical properties were related to the in vitro bioactivity. We believe that this approach can form a novel formulation strategy for the design of particulate, chitosan based siRNA formulations devoid of large excess use of chitosan (as free protusions or as excipient
Conclusions
In conclusion, chitosans derived from fungal source allowed for the generation of low N:P chitosan/siRNA nanoparticles with good bioactivity and minimal impact on cell viability with siRNA concentrations ranging from 37 to 150 nM. Furthermore this study is providing important insight into the formation of siRNA/chitosan polyplexes by rigorous exploitation of thermodynamic methods and imaging techniques. Correlation of these results with biological performance led to the conclusion that although
Acknowledgments
We are very grateful to Drs F.A. Asselbergs, W. Wishart and R. Widmer, A. Dattler, S. Haas and C. Cantina for support with the in vitro cell cultures; Dr. Eckes for assistance in image analysis, Dr. Rausch for assistance with the confocal microscopy; Prof. Dr. H. Stahlberg, Drs M. Chami and K. rGoldie from C-CINA, Biozentrum University of Basel for electron microscopy pictures, Prof. Dr. Wagner from the Ludwig Maximillians University, Munich; We would especially like to thank Dr. Giulio
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