Table 1.

Insoluble chitosan microparticles: Published examples of in vivo biomedical use, and in vitro investigation of cell responses.

Method of insoluble chitosan microparticle preparation	Chitosans analyzed	Application
Neutral precipitation of acid-soluble chitosan in vivo or in vitro
Mix acid-soluble chitosan solution or chitosan-glycerol phosphate solution pH 4–6.8 with whole blood and/or directly apply to bleeding tissues	80% DDA 75%–82% DDA, 150–250 kDa	Hemostatic in vivo [17] Bone and articular cartilage repair in vivo [20]
Inject mixtures of chitosan-DNA, with a molar excess of chitosan, in intramuscular, subcutaneous sites	92% DDA, 10 kDa 80% DDA, 10 kDa 80% DDA, 80 kDa	Gene delivery in vivo [24]
Combine soluble chitosan at 5- to 10-fold molar excess with DNA, pipette into cell culture medium DMEM+10% serum pH 7.6	80% DDA, 15 kDa 92% DDA, >100 kDa 94% DDA, 52 kDa	In vitro DNA delivery: A549, Hela, B16 cells, HEK293 cells [25,26]
Pipette acid-soluble chitosan pH 5.0 into basal media pH 7.6 (DMEM, αMEM, RPMI±10% to 16% fetal bovine serum)	80% DDA, 179 kDa 81% DDA, 35 kDa 80% DDA, 179 kDa 95% DDA, 168 kDa 92% DDA, 10 kDa	In vitro bone marrow stromal cell osteogenesis [27] In vitro macrophage activation [28] In vitro neutrophil chemotaxis, degranulation, chitosan uptake [14] In vitro chitosan-HEK293 cell adsorption/uptake [26]
Pre-formed chitosan microparticles
Pre-formed microparticles (glutaraldehyde and Tween surfactant) injected into the mouse foot pad or added to DMEM+heat-inactivated 10% fetal bovine serum	75%–85% DDA, 164 kDa	Vaccine, lymph node trafficking in vivo [29] In vitro HEK293, A549, RAW264 cell uptake [29]
1 µm or 3.5 µm pre-formed chitosan microparticles added to RPMI+10% serum, pH 7.2	≥80% DDA	Wound-repair applications: in vitro neutrophil chemotaxis [30] in vitro macrophage activation [31]