There are many ways to prepare chitosan nanoparticles. But, the Ionic Gelation Method (also called ionotropic gelation) has been widely researched and the simplest to perform. Its also very cost-effective from bench to commercial scale-up. This brief review covers its advantages and disadvantages and the steps for preforming the method.

Chitosan is a polysaccharide polymer that’s biocompatible and biodegradable, extracellular, and holds a positive charge. These are a few of chitosan’s features which makes it flexible for formulating a variety of materials as solutions for a variety of applications. Here are only a few examples:

Controlled drug delivery,  Tissue-engineering, Woundcare, Biosensors Membrane separators Antibacterial coatings, etc.

Formulating chitosan in nanoparticles enhances its ability to dissolve, entrap, encapsulate, and/or have bioactive agents attach to its nanoparticle matrix. These systems have high surface areas where bioactives can absorb on their surface area. Their nanometer-size also promotes effective permeation through cell membranes and stability in the bloodstream. Chitosan nanoparticles can also carry low to high molecule weight and negatively charged drugs, proteins and DNA to target cells, tissues, and organs. These features and functionalities also make chitosan nanoparticles suitable for parenteral and mucosal routes of administration, i.e., oral, nasal, and ocular mucosa.

To achieve these functionalities, chitosan nanoparticles must be prepared with methods that consider implementation of the technique and the results it can provide to the application in question.  With this, the Ionotropic Gelation Method is one of the most commonly used methods to prepare chitosan nanoparticles because it’s:

• An relatively easy technique to perform
• Conducted under mild conditions and requires less harsh solvents
• Easily modified and controllable.

Chitosan nanoparticles preparation by ionic gelation is based on chitosan having a high degree of protonation of the amine functions and the ability to form hydrogels in presence of specific polyanions.  When chitosan is cross-linked with an agent, such as sodium tripolyphosphate (TPP), their opposing charges causes inter- and intra- molecular cross-linkages and produces chitosan nanoparticles.  This process is known as the Ionic Gelation Method for producing chitosan nanoparticles.^1,2

Ionotropic gelation is based on the capability of polyelectrolytes to cross-link in the presence of counter ions to form nanoparticles.⁴

In the ionic gelation method, chitosan polysaccharide is dissolved in aqueous acidic solution to obtain the cation of chitosan. This solution is then added under continuous stirring to polyanionic tripolyphosphate solution. Due to the complexation between opposing charges (positive/negative), chitosan undergoes ionic gelation and precipitates to form spherical particles.  The following are the steps to prepare chitosan nanoparticles with the ionic gelation method;

There are several methods for preparing chitosan nanoparticles based on the spontaneous formation of complexes between the polysaccharide chitosan and various polyanions.⁷

(a) Low-molecular weight poly-anions (e.g., sodium pyrophosphate, tripolyphosphate, tetrapolyphosphate, octapolyphosphate, hexametaphosphate)
(b) Hydrophobic polyanions (e.g., sodium alginate and k-carrageenan)
(c) High-molecular weight ions (e.g., octyl sulfate, dodecyl sulfate, hexadecyl sulfate, and cetyl-stearyl sulfate)

A number of factors affect chitosan nanoparticles characteristics following its preparation with the ionic gelation method. These factors have been studied across several studies and have established a relation between key preparation variables (e.g. pH, concentration, ratios of components, method of mixing, etc.)  and the characteristics and behaviors of chitosan nanoparticles (particle size, zeta potential, drug release behavior, etc.).^8,9,10 

Parameters that affect the structure and behavior of Chitosan Nanoparticles obtained from the ionic gelation method:

Procedures;

• Stirring pattern (speed and type of vessel)
• Centrifugation (speed and duration)⁶

 Optimizing variables include;

• Concentration and mass ratios of materials¹¹
• Temperature of the reacting solutions⁶

Examples of influencing factors; 

• Both the concentration of acetic acid used to dissolve chitosan and the temperature used during the cross-linking process significantly affects the polydispersity of the chitosan/TPP nanoparticles.¹²

• Molecular Weight (Mw) and the Degree of Deacetylation (DDA%) are key characteristics found to affect the particle size and surface charge of chitosan NPs prepared by ionic gelation.

• Chitosan nanoparticles modified the mechanism of cellular uptake but did not change the cytotoxicity of the polymer toward A549 cells. Chitosan Degree of Deacetylation (DDA%) had a greater influence than Molecular Weight (Mw) on the uptake and cytotoxicity of chitosan nanoparticles because of its effect on the zeta potential of the nanoparticles.¹³

• Its reported that a high degree of deacetylated chitosan with a narrow molecular weight distribution proved vital for controlling its particle size distribution when producing chitosan nanoparticles by ionic gelation and using chitosan and TPP.¹⁴

• Ionic interactions between chitosan and TTP are dependent on the charge density of the chitosan/TPP and solution.

• pH can be controlled and tuned by the pH of the chitosan/TTP solution.

• Accompanying materials used in the chitosan nanoparticle matrix have significant affect(s) on the final chitosan characteristics and behaviors.

• Tripolyphosphate (TPP) is often a cross-linking of choice in preparing chitosan nanoparticles as TPP is nontoxic, multivalent, and forms gels through ionic interactions. The production of nanoparticles by ionic gelation results in smaller particles the higher the amounts of cross-linker¹⁵

• Nanoparticles are readily formed due to complexation between positive and negative charged species during stirring at room temperature, resulting in separation of chitosan in spherical particles of different sizes and surface charges. The size and surface charge of the particles can be optimized by adjusting the ratio of chitosan and stabilizer.¹²

• The size and surface charge of particles can be modified by varying the ratio of chitosan and stabilizer.¹²

• The encapsulation efficiency was highly decreased by the increase of initial Bovine serum albumin (BSA) and chitosan concentration; higher loading capacity of BSA sped up the BSA release from the nanoparticles. Adding PEG hindered the BSA encapsulation and accelerated the release rate.¹⁶

• Volume of resuspension has been reported to have an effect on.¹⁴

• The type of chitosan used in preparing nanoparticles with the ionic gelation method has also been reported to have an effect. Chitosans are available in different chemical structures either as a base or a salt, e.g., chloride, lactate, glutamate, etc.

• It has been reported that the relationship between free amino groups on the surface and the characteristics of chitosan nanoparticles prepared by the ionic gelation method were not affected by TPP concentration in certain cases.¹⁷

• Easy Process to Conduct – Simple steps
• Mild processing conditions – an aqueous environment low shear forces, no heat^18,19
• Economical
• Avoids Harsh Organic Solvents – Reversible physical cross-linking by electrostatic interaction can replace chemical cross-linking to avoid reagents’ risk of toxicity¹⁷
• Controllable and Tunable
• Nontoxic – with low probability of altering the chemistry of the underlying drug or therapeutic agent²⁰
• Facilitates low molecular weight drugs²¹

• Poor stability in acidic conditions
• Difficult in entrapping high molecular weight drugs
• TPP/CS nanoparticles  usually possess poor mechanical strength
• Technology transfer to scale up to commercial production is very difficult.

                                                                          References for disadvantages:  22,23,24

An ever growing number of research studies demonstrate that chitosan nanoparticles and nanostructures are effective in encapsulating, immobilizing, controlling, and delivering bioactive therapeutic ingredients.  It can do so via various routes of administration (e.g., parenteral, oral, nasal, ocular mucosa, implanted, etc.) and targeted to various organs, tissues, and cells.   To accomplish all this chitosan and its nanoparticles are best produced with methods that are for the most part easy to perform, economical, non-toxic, and renders a system that is controllable and tunable; Ionic (Ionotropic) Gelation is one such method.

References

1. Janes, Kevin A. et al., Chitosan nanoparticles as delivery systems for doxorubicin, Journal of Controlled Release Volume 73, Issues 2–3, 15 June 2001, Pages 255-267.  [CrossRef] [PubMed]
2. Muzzarelli, RAA. Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids.  Carbohydrate Polymers Volume 77, Issue 1, 22 May 2009, Pages 1-9.  [CrossRef]
3. Bhattarai, N.  Chitosan-based hydrogels for controlled, localized drug delivery.  Adv Drug Deliv Rev. 2010 Jan 31;62(1):83-99).  [PubMed]
4. Patil, JS.  Ionotropic Gelation and Polyelectrolyte Complexation: The Novel Techniques to Design Hydrogels Particulate Sustained, Modulated Drug Delivery System: A Review.  Digest Journal of Nanomaterials and Biostructures Vol. 5, No 1, March 2010, p. 241 – 248, (p242). [CrossRef]
5. Ur-Rehman, T et al. Chitosan in situ gelation for improved drug loading and retention in poloxamer 407 gels.  Int J Pharm. 2011 May 16;409(1-2):19-29.  [CrossRef] [PubMed]
6. Carvalho, et al.  Mucosal delivery of liposome-chitosan nanoparticle complexes. Methods Enzymol. 2009;465:289-312. [CrossRef] [PubMed]
7. Calvo, P. et al.  Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers.  J. Appl. Polym. Sci. 1997, 63(1) 125-132.  [CrossRef] [PubMed]
8. Al-Qadi, S. et al. Microencapsulated chitosan nanoparticles for pulmonary protein delivery: in vivo evaluation of insulin-loaded formulations. J Control Release. 2012 Feb 10;157(3):383-90.  [CrossRef] [PubMed]
9. Bonferoni, MC.  Chitosan and its salts for mucosal and transmucosal delivery. Expert Opin Drug Deliv. 2009 Sep;6(9):923-39.  [PubMed]
10. Nasti, Alessandro, et al.  Chitosan/TPP and Chitosan/TPP-hyaluronic Acid Nanoparticles: Systematic Optimisation of the Preparative Process and Preliminary Biological Evaluation. Pharmaceutical research. 26. 1918-30. 10.1007/s11095-009-9908-0.  [CrossRef]  [PubMed]
11. Calvo, P. et al.  Novel hydrophilic chitosan- polyethylene oxide nanoparticles as protein carriers.  J. Appl. Polym. Sci. 1997, 63(1) 125-132.  [CrossRef]  [PubMed]
12. Fan, W. Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique. Colloids Surf B Biointerfaces. 2012 Feb 1;90:21-7.  [CrossRef] [PubMed]
13. Huang, M et al.  Uptake and cytotoxicity of chitosan molecules and nanoparticles: effects of molecular weight and degree of deacetylation. Pharm Res. 2004 Feb;21(2):344-53.  [CrossRef] [PubMed]
14. Zhang, H et al.  Monodisperse chitosan nanoparticles for mucosal drug delivery. Biomacromolecules. 2004 Nov-Dec;5(6):2461-8.  [PubMed]
15. Grenha, A. et al. Microencapsulated chitosan nanoparticles for lung protein delivery. Eur J Pharm Sci. 2005 Jul-Aug;25(4-5):427-37.  [CrossRef] [PubMed]
16. Xu Y, Du Y.  Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles. Int J Pharm. 2003 Jan 2;250(1):215-26.  [CrossRef] [PubMed]
17. Lin, AH.  Free amino groups on the surface of chitosan nanoparticles and its characteristics.  Yao Xue Xue Bao. 2007 Mar;42(3):323-8.  [PubMed]
18. Agnihotri, SA. Recent advances on chitosan-based micro- and nanoparticles in drug delivery.  J Control Release. 2004 Nov 5;100(1):5-28.  [CrossRef] [PubMed]
19. Tiyaboonchai, W.  Chitosan Nanoparticles : A Promising System for Drug Delivery.  Naresuan University Journal 2003; 11(3): 51-66 51.  [CrossRef]
20. Park, JS. et al.  N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs: endocytosis, exocytosis and drug release.  J Control Release. 2006 Sep 28;115(1):37-45. Epub 2006 Jul 20.  [CrossRef] [PubMed]
21. Vila, A. et al.  Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice. Eur J Pharm Biopharm. 2004 Jan;57(1):123-31.  [CrossRef] [PubMed]
22. Nagpal, K. et al.  Chitosan nanoparticles: a promising system in novel drug delivery. Chem Pharm Bull (Tokyo). 2010 Nov;58(11):1423-30.  [CrossRef] [PubMed]
23. Gonçalves, IC et al.  The potential utility of chitosan micro/nanoparticles in the treatment of gastric infection.  Expert Rev Anti Infect Ther. 2014 Aug;12(8):981-92.  [CrossRef] [PubMed]
24. Vila, A. et al.  Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice.  Eur J Pharm Biopharm. 2004 Jan;57(1):123-31.  [CrossRef] [PubMed]