Fabrication of porous chitosan particles using a novel two-step porogen leaching and lyophilization method with the label-free multivariate spectral assessment of live adhered cells

Colloids Surf B Biointerfaces. 2021 Dec;208:112094. doi: 10.1016/j.colsurfb.2021.112094. Epub 2021 Sep 1.


Porous chitosan (CS) particles were fabricated using a novel two-step technique that employed a porogen leaching phase followed by lyophilization or freeze-drying. Poly(ethylene glycol) (PEG) was mixed as a porogen in two different quantities with the CS solution before particle synthesis via coacervation. After the PEG leached out into deionized (DI) water at an elevated constant temperature, the final freeze-dried CS particles revealed surface features that resembled pore pockets. A three-dimensional (3D) culture of murine osteoblast cell line (OB-6) was seeded on these particles to analyze the effect of the porous structure on the cell activity, as compared to a control group with no added porogen. The results highlighted an enhancement in cell adhesion and proliferation on the two porous sample groups. A Raman spectroscopy-based label-free technique for live cell biomarker analysis was applied using multivariate spectral analysis. Results of the spectral analysis in the molecular fingerprint region corresponding to the Raman shift between 900 cm-1 and 1700 cm-1inferred inter-group variations. The bands at 1005 cm-1 and 1375 cm-1 were assigned to the live cell biomarkers phenylalanine and glycosaminoglycan, respectively, and were assessed during the multivariate spectral analysis. The corresponding score plot and loading information generated from the Principal Component Analysis (PCA) of the Raman spectrum at day 7 and day 14, pointed at inter-group spectral variations related to cell adhesion and proliferation between the two porous CS particle groups and the control.

Keywords: Label-free Raman; Lyophilization; Porogen leaching; Porous particle; Principal component analysis.

MeSH terms

  • Animals
  • Cell Adhesion
  • Chitosan*
  • Freeze Drying
  • Mice
  • Porosity
  • Tissue Scaffolds


  • Chitosan