Formation and dissolution of chitosan/pyrophosphate nanoparticles: Is the ionic crosslinking of chitosan reversible?

https://doi.org/10.1016/j.colsurfb.2013.11.032Get rights and content

Highlights

  • Ionically crosslinked chitosan/pyrophosphate (PPi) particles were investigated.

  • Their size could be predictably tuned by varying the chitosan concentration.

  • Chitosan/PPi particles were colloidally stable even at high PPi concentrations.

  • Their formation/dissolution cycle exhibited hysteresis.

  • The irreversibility of their formation was modeled using the Bragg–Williams theory.

Abstract

Ionically crosslinked chitosan particles with submicron dimensions attract widespread interest as materials for controlled release. To this end, we have examined the formation and dissolution of nanoparticles prepared by crosslinking chitosan with pyrophosphate (PPi). The formation of these particles required a critical PPi concentration (which increased with the chitosan concentration), and their z-average hydrodynamic diameters could be predictably tuned from roughly 60 to 220 nm by varying the concentration of the parent chitosan solutions. Unlike the nanoparticles crosslinked with the commonly used tripolyphosphate (TPP), which coagulated and precipitated when TPP was in excess, the chitosan/PPi nanoparticles remained colloidally stable even at high PPi concentrations. Moreover, the analysis of their dissolution revealed hysteresis in the particle formation/dissolution cycle, where portions of the crosslinked chitosan/PPi complexes remained stably intact at PPi concentrations below those required for their formation. This irreversible behavior was surmised to reflect the cooperativity of chitosan/PPi binding and was qualitatively modeled using the Bragg–Williams theory.

Introduction

Ionically crosslinked chitosan particles with submicron dimensions are widely studied as materials for the delivery of drugs, genes and food additives [1], [2], [3], [4]. They are typically prepared by mixing multivalent anions, such as tripolyphosphate (TPP), with dilute chitosan solutions (see Figs. 1a and 1b), which leads to spontaneous micro- and nanoparticle formation. These colloidal carriers provide multiple advantages, such as forming under very mild conditions [5], being biocompatible and mucoadhesive [5], [6], [7], [8], enhancing drug penetration across epithelial membranes [9], [10], and stabilizing proteins against denaturation [11].

Despite the numerous studies exploring their preparation and use [1], [2], [3], [4], [5], [6], [9], [10], [12], [13], work with these materials has mostly been limited to particles formed using TPP as the crosslinker. While recently there has been some interest in submicron particles prepared using other ionic crosslinkers (e.g., pyrophosphate (PPi) (Fig. 1c) [14], [15] and sulfate [16]), factors controlling their formation, structure and stability have not been studied in detail. Therefore, although these alternative crosslinkers could yield a broader range of desirable particle properties, there is a dearth of guidelines for their use.

Furthermore, while much attention has been devoted to studying particle formation and size distributions [1], [3], [12], [13], [17], little has been done to investigate the dissociation of ionically crosslinked chitosan particles. When ionically crosslinked particles are placed into an excess of crosslinker-free solution (i.e., during their application) it is probable that the ionic crosslinker is leached from the particles, causing the particles to dissolve. Yet, although several studies have examined the shelf life of chitosan/TPP particles [18], [19], [20], and it is known that chitosan crosslinked with TPP or PPi dissolves in the limit of low pH (e.g., pH 1, where the crosslinking anions become protonated) [21], [22], the dissolution of ionically crosslinked chitosan through simple crosslinker leaching (achieved without pH reduction) remains virtually unexplored. Indeed, the only such work known to us was a protein uptake/release study where the structure of protein-laden chitosan/TPP particles became less dense during in vitro protein release experiments [23]. Due to the presence of protein, however, the role of crosslinker leaching in these structural changes (versus that of protein elution) remains unclear.

To begin addressing these issues, the present study had two objectives: (1) to provide systematic guidelines for the preparation of chitosan/PPi nanoparticles which, as shown herein, behave dissimilarly from those prepared using TPP; and (2) to investigate the dissolution of ionically crosslinked chitosan particles. This second objective was pursued using PPi instead of the more-common TPP because chitosan/PPi binding was much weaker than chitosan/TPP binding [14]. In other words, because the very strong chitosan/TPP binding made TPP elution challenging to achieve [22], the PPi-based particles provided a more-tractable experimental system. Based on these considerations, this study consisted of three parts. First, dynamic and electrophoretic light scattering, scanning transmission electron microscopy (STEM), stopped-flow turbidimetry and viscometry were used to investigate the compositional parameters affecting the formation, structure and colloidal stability of chitosan/PPi nanoparticles. Second, light scattering was used to investigate particle dissolution that occurred when PPi was leached from the complex. Third, the irreversible formation/dissolution behavior revealed by these experiments was qualitatively modeled using the Bragg–Williams theory.

Section snippets

Materials and methods

Low molecular weight chitosan (nominal MW = 50–190 kDa), sodium pyrophosphate (Na2PO4) and acetic acid were purchased from Sigma–Aldrich (St. Louis, MO) and used as received. The degree of chitosan deacetylation was 90% [24]. All experiments were performed using 18.2  m Millipore Direct-Q 3 deionized water at pH 4.0 (using acetic acid to adjust the pH) and, unless otherwise noted, at room temperature.

To prepare the particles, 10 mL of 0.01–0.20 wt% chitosan solution (0.55–11 mM in its cationic

Formation of chitosan/PPi particles

The formation of chitosan/PPi particles was determined from the changes in light scattering intensity during the titration of PPi into chitosan solutions. At low PPi concentrations (where no particles formed) the light scattering intensity remained similar to that of PPi-free chitosan solutions. At the onset of particle formation, however, the scattering intensity began to suddenly increase (see Fig. 2a). Finally, at even higher PPi concentrations, where the particle formation process ended,

Conclusion

The crosslinking of chitosan with PPi yields nanoparticles, whose size can be predictably tuned by varying the concentration of the parent chitosan solutions. The formation of these particles requires a critical PPi concentration, below which no particles form. Above this critical concentration, chitosan chains begin to self-assemble into colloidal complexes. The kinetics of this process depend strongly on the ionic crosslinker concentration. Unlike the more common chitosan/TPP particles (which

Acknowledgements

We are grateful to the National Science Foundation (CBET-1133795) for supporting this work, Udaka K. de Silva for assistance with the light scattering experiments, and Yan Huang for helpful discussions and assistance with STEM.

References (38)

  • K.A. Janes et al.

    Adv. Drug Deliv. Rev.

    (2001)
  • M. Garcia-Fuentes et al.

    J. Control. Release

    (2012)
  • H. Takeuchi et al.

    Adv. Drug Deliv. Rev.

    (2001)
  • D. Vllasaliu et al.

    Int. J. Pharm.

    (2010)
  • X. Wang et al.

    Eur. J. Pharm. Biopharm.

    (2008)
  • W. Fan et al.

    Colloids Surf. B

    (2012)
  • O. Borges et al.

    Int. J. Pharm.

    (2005)
  • Q. Gan et al.

    Colloids Surf. B

    (2005)
  • T. Lopez-Leon et al.

    J. Colloid Interface Sci.

    (2005)
  • G.A. Morris et al.

    Carbohydr. Polym.

    (2011)
  • M.L. Tsai et al.

    Carbohydr. Polym.

    (2011)
  • X.Z. Shu et al.

    Eur. J. Pharm. Biopharm.

    (2002)
  • Q. Gan et al.

    Colloids Surf. B

    (2007)
  • W. Fan et al.

    Colloids Surf. B

    (2012)
  • P. Calvo et al.

    J. Appl. Polym. Sci.

    (1997)
  • B. Hu et al.

    J. Agric. Food Chem.

    (2008)
  • K.A. Janes et al.

    J. Appl. Polym. Sci.

    (2003)
  • H. Zhang et al.

    Biomacromolecules

    (2006)
  • I.A. Sogias et al.

    Biomacromolecules

    (2008)
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