Elsevier

Journal of Controlled Release

Volume 157, Issue 2, 30 January 2012, Pages 297-304
Journal of Controlled Release

Biophysical properties of chitosan/siRNA polyplexes: Profiling the polymer/siRNA interactions and bioactivity

https://doi.org/10.1016/j.jconrel.2011.08.023Get rights and content

Abstract

Chitosans are naturally occurring polymers widely used in life science to mediate intracellular uptake of nucleic acids such as siRNA. Four chitosans of fungal origin (Agaricus bisporus; molecular weights MW = 44, 63, 93 and 143 kDa) were used in this study and profiled for size, viscosity and hydrodynamic radius using gel permeation chromatography (GPC). Polyplexes made of these chitosans and siRNA were developed and optimized for transfection efficacy in vitro. The characteristics of these polyplexes were low chitosan:siRNA ratios (4–8; N:P) similar positive zeta potential (20–30 mV) and comparable particle sizes (about 150 nm). Endogenous luciferase reporter gene down-regulation in human epithelial H1299 cells at nanomolar concentrations (37.5–150 nM) was significantly stronger for the lower molecular weight chitosans. The impact of these low N:P polyplexes on the cellular viability was minimal also at 150 nM. To help develop an understanding of these differences, an energetic profile of the molecular interactions and polyplex formation was established by isothermal titration calorimetry (ITC). The four polyplexes exhibited strong binding enthalpies delta Hbind(− 84 to −102 kcal/mol) resulting in nanomolar dissociation constants. Intracellular trafficking studies using rhodamine labeled siRNA revealed that polyplexes made from smaller MW chitosans exhibited faster cellular uptake kinetics than their higher MW counterpart. Transmission electron microscopy and small angle X-ray scattering studies (SAXS) revealed that the 44 kDa derived polyplexes exhibited regular spherical structure, whereas the 143 kDa chitosan polyplex was rather irregularly shaped. With regards to adverse effects these low N:P chitosan/siRNA formulations represent an interesting alternative to so far reported chitosan polyplexes that used vast N:P excess to achieve similar 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.

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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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