TABLE 2.

Articles on chitosan nanoparticles without TPP as crosslinker, main findings.

Chitosan characteristics	Nanoparticle composition	Active ingredient	Cell line/species	Toxicity assay	Cytotoxicity results	References
Chitosan DDA 95%	Chitosan/Na2SO4 and Chitosan/Na2SO4/alginate	Ovalbumin	Spleen cells from female BALB/c mice	MTT assay Trypan blue PI stain	No cytotoxicity was detected for any of the unloaded NPs (0.28 and 0.42 mg/ml), on the contrary increased proliferation was observed	Borges et al. (2006)
Chitosan low MW DDA 95%	Chitosan/Na2SO4	C48/80 (mast cell activator)	Spleen cells from C57BL/6 mice A549 cell line	MTT assay	Spleen cells: >80% cell viability when incubated with unloaded NPs ≤1.08 mg/ml. A549 cell line: >70% cell viability when incubated with unloaded NPs ≤1.5 mg/ml	Bento et al. (2019)
Chitosan 71.3 kDa DDA 80%	Chitosan/Na2SO4	Newcastle disease virus F gene DNA (pFNDV)	SPF chickens 293-T cells (chicken embryo kidney cellsCEK cells)	Safety test WST-8 kit	Intranasal administration of pFNDV loaded-NPs considered safe. 84% survival rate of kidney cells, no significant changes in cell morphology	Zhao et al. (2014)
Chitosan 200–300 kDa DDA 85%	Chitosan/mucin	Insulin	Wistar rats	Liver enzymes MTT assay	In vivo: No significant change in liver enzymes was seen after 3 days of orally administrated 50 IU/kg unloaded NPs.	Mumuni et al. (2020)
					In vitro: 0–500 μg/ml showed >98% cell viability after 24 h.
Chitosan	Chitosan	Chitosan Thymol (2-isopropyl-5- ethylphenol	Nile tilapia fingerlings	Biochemical Macroscopic Histologic	No significant change in survival rate between experimental groups when fed with unloaded and Thymol-loaded NPs.	Abd El-Naby et al. (2020)