Abstract
This study is about linking preparative processes of nanoparticles with the morphology of the nanoparticles and with their efficiency in delivering payloads intracellularly. The nanoparticles are composed of hyaluronic acid (HA) and chitosan; the former can address a nanoparticle to cell surface receptors such as CD44, the second allows both for entrapment of nucleic acids and for an endosomolytic activity that facilitates their liberation in the cytoplasm. Here, we have systematically compared nanoparticles prepared either A) through a two-step process based on intermediate (template) particles produced via ionotropic gelation of chitosan with triphosphate (TPP), which are then incubated with HA, or B) through direct polyelectrolyte complexation of chitosan and HA. Here we demonstrate that HA is capable to quantitatively replace TPP in the template process and significant aggregation takes place during the TPP–HA exchange. The templated chitosan/HA nanoparticles therefore have a mildly larger size (measured by dynamic light scattering alone or by field flow fractionation coupled to static or dynamic light scattering), and above all a higher aspect ratio (Rg/RH) and a lower fractal dimension. We then compared the kinetics of uptake and the (antiluciferase) siRNA delivery performance in murine RAW 264.7 macrophages and in human HCT-116 colorectal tumor cells. The preparative method (and therefore the internal particle morphology) had little effect on the uptake kinetics and no statistically relevant influence on silencing (templated particles often showing a lower silencing). Cell-specific factors, on the contrary, overwhelmingly determined the efficacy of the carriers, with, e.g., those containing low-MW chitosan performing better in macrophages and those with high-MW chitosan in HCT-116.
Highlights
Chitosan is a linear copolymer of β-1,4-ᴅ-glucose-2-amine and N-acetyl-ᴅ-glucose-2-amine, and is commonly employed as the cationic component in polyplexes and other drug delivery vehicles [1,2,3]
The distinction between the two is subtle, since they are based on a common driving force, i.e., the electrostatic attraction between protonated amines on chitosan and multiply charged anions, which effectively act as cross-linkers
The stability of the nanoparticles was similar: the behaviour of the nanoparticles prepared by the direct method upon dialysis, storage and dispersion in different media (Supporting Information File 1, Section SI2 and Figure S1) was comparable to that previously reported by our group for the templated method [18]
Summary
Chitosan is a linear copolymer of β-1,4-ᴅ-glucose-2-amine and N-acetyl-ᴅ-glucose-2-amine, and is commonly employed as the cationic component in polyplexes and other drug delivery vehicles [1,2,3]. In comparison to other polycations, its main advantages are the low toxicity and its biodegradability. Biodegradation can occur both enzymatically and oxidatively [4]. A number of methods can be employed to prepare chitosan-based nanoparticles [5,6,7,8], the most popular being ionotropic gelation and polyelectrolyte complexation. The distinction between the two is subtle, since they are based on a common driving force, i.e., the electrostatic attraction between protonated amines on chitosan and multiply charged anions, which effectively act as cross-linkers. Electrostatic interactions may be used to hold together a particle, and to encapsulate and retain payloads such as nucleic acids (either in combination with other anionic components [9], or as the only negatively charged molecule [10])
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