TABLE 4.

Comparative analysis regarding the attributes of different nanotoxicity evaluation modalities.

Attributes	2D monolayer culture model	3D organoid model	Lab animal model
Ethical issues	No ethical restriction	Lucid ethical restriction, only limited ethical issues arise pertaining to stem cell research and stem cell therapies	Stringent adherence to the ethical guidelines for animal experimentation is compulsory
Economics of operation and maintains	Least	Moderate to high depending upon the experimental requirements	Resource intensive
Batch variation in replicates	Least batch variation under predefined experimental set up	Low to moderate batch variation depending upon the matrix material and customization protocol	Moderate to high individual variation based upon the pathophysiological and nutritional status of the animals
Survival	Survival in days thus unsuitable for long-term toxicity analysis	Moderate lifespan, usually up to few months which can be enhanced by vascularization	Enough lifespan, even suitable as chronic toxicity assessment model
Efficiency to mimic the real in vivo condition of the target species	Very limited as it is devoid of spatial architecture, immune system, and communication machinery, etc.	Considerably efficient to mimic the near-physiological microenvironment, possess several structural and functional attributes of the real target organ, most importantly organoid from the target species or patient-derived organoid can be used to nullify interspecies variations in drug metabolism, even suitable for developing personalized medicine	Provides real in vivo condition but substantial interspecies anatomical and metabolic variations, particularly in drug metabolism, diversity in omics attributes often yields false prediction in the targeted species
Feasibility for structural and functional integrity study	Least	Optimum	Comprehensive
Scope for drug penetration and biodistribution analysis	Very limited to none	Multilayered organoids provide ample opportunity for drug penetration and biodistribution analysis	Most suitable model for such requirement
Cellular heterogeneity	Minimal to negligible	Considerable cellular heterogeneity is present	Extensive
Level of cell-to-cell interaction	Minimal	Optimum	Comprehensive
Tissue-native immune system interaction	None	Optimum	Extensive
Scope for organ–microenvironment interaction	None	Optimum and can be regulated depending upon the requirement	Comprehensive and regulated
Feasibility for organ–organ interaction	None	Not possible for organoid recapitulating a single-type organ, but possible in multi-organoid-on-a-chip microfluidic platform or co-customized multiple organoid system connected by vasculature/luminal organoid	Extensive
Cell-blood vessel interaction	None	Only in vascularized organoid	Yes
Fluid flow perfusion	No	Only in vascularized organoids or organoid-on-a-chip microfluidic platform or organoids connected by 3D-bioprinted lumens	Yes
Deposition toxicity issue of nano-drugs	No deposition toxicity issue of nanoparticles arise	Minimum possibility of deposition toxicity for testing the nano-drugs	Frequent probability of deposition toxicity for testing the nano-drugs
Patient-specific model/personalized medicine	Partial using specific cell lines	Most appropriate in vitro model	Very limited to none