Table 3:

Biological characterization methods for CPs

Technique	Purpose
Endotoxin assays (Limulus Amebocyte Lysate Assay)	Confirm absence of contamination with endotoxin.
In vitro release profiles	Determine pH/buffer dependence and stability.
In vitro cytotoxicity assay (MTT, MTS, CCK-8, LDH, TUNEL) and IC50	Determine toxicity to most relevant cell types.
Confocal microscopy	Observe cellular association and uptake.
Flow cytometry	Observe cellular association. When paired with antigen recall, can evaluate specific cellular markers that represent long-term immunity or activation markers.
ELISA, Luminex®, and ELISpot	Measure cytokine production from cells treated in vitro. Determine activation of key cell populations necessary for vaccination and assessment of cellular response when paired with antigen recall. Measure antibodies from serum for humoral response.
Hematoxylin and eosin histology staining and hemolysis	Determine toxicity in animal models.
In vivo imaging systems	Tracking the biodistribution of material upon in vivo administration.
Challenge studies	Evaluation of the material’s ability to protect against lethal disease.