Protein cytoplasmic delivery using polyampholyte nanoparticles and freeze concentration

Biomaterials. 2014 Aug;35(24):6508-18. doi: 10.1016/j.biomaterials.2014.04.030. Epub 2014 May 10.


A protein delivery method using freeze concentration was presented with a variety of polyampholyte nanocarriers. In order to develop protein nanocarriers, hydrophobically modified polyampholytes were synthesized by the succinylation of ε-poly-l-lysine with dodecyl succinic anhydride and succinic anhydride. The self-assembled polyampholyte aggregated form nanoparticles through intermolecular hydrophobic and electrostatic interactions when dissolved in aqueous media. The cationic and anionic nanoparticles were easily prepared by changing the succinylation ratio. Anionic or cationic proteins were adsorbed on/into the nanoparticles depending on their surface charges. The protein-loaded nanoparticles were stable for at least 7 d. When L929 cells were frozen with the protein-loaded nanoparticles in the presence of a cryoprotectant, the adsorption of the protein-loaded nanoparticles was enhanced and can be explained by the freeze concentration mechanism. After thawing, proteins were internalized into cells via endocytosis. This was the first report that showed that the efficacy of protein delivery was successfully enhanced by the freeze concentration method. This method could be useful for in vitro cytoplasmic protein or peptide delivery to various cells for immunotherapy or phenotype transformations.

Keywords: Freeze concentration; Nanoparticles; Polyampholytes; Protein delivery.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adsorption
  • Animals
  • Buffers
  • Cattle
  • Cell Survival / drug effects
  • Cryopreservation
  • Cryoprotective Agents / pharmacology
  • Cytoplasm / drug effects
  • Cytoplasm / metabolism*
  • Drug Delivery Systems*
  • Endocytosis / drug effects
  • Freezing*
  • Hydrophobic and Hydrophilic Interactions
  • Magnetic Resonance Spectroscopy
  • Muramidase / metabolism*
  • Nanoparticles / chemistry*
  • Nanoparticles / ultrastructure
  • Particle Size
  • Serum Albumin, Bovine / metabolism*
  • Static Electricity


  • Buffers
  • Cryoprotective Agents
  • Serum Albumin, Bovine
  • Muramidase