Table 3.

Overview of relevance and major challenges related to measurement of drug loading and release with reference to regulatory documents

Area	Subcategory	Relevance	Challenges	Gaps
Drug loading & release [[6], [7], [8], [9], [10], [11], [12]]	1) Total drug loading	Impact on efficacy safety Monitoring of API leakage during storage and use (stability)	•Need to adapt the methods to each nanocarrier/API combination •Separation of API from nanocarrier prior to API quantification (subcategory 2) can induce artefacts	detection and quantification of large API such as nucleic acids
	2) Free vs encapsulated drug