Enhanced Adsorption of a Protein–Nanocarrier Complex onto Cell Membranes through a High Freeze Concentration by a Polyampholyte Cryoprotectant

Abstract

The transportation of biomolecules into cells is of great importance in tissue engineering and as stimulation for antitumor immune cells. Previous freezing strategies at ultracold temperatures (-80 °C) used for intracellular transportation exhibit certain limitations such as extended time requirements and harsh delivery system conditions. Thus, the need remains to develop simplified methods for safe nanomaterial delivery. Here, we demonstrated a unique strategy based on the ice-crystallization-induced freeze concentration for protein intracellular delivery in combination with a polyampholyte cryoprotectant. We found that upon sustained lowering of the temperature from -6 to -20 °C over a short duration, the adsorption of proteins onto the peripheral cell membrane was markedly increased through the facile ice-crystallization-induced freeze concentration. Furthermore, we proposed a freeze concentration factor (α) that depends on the freezing-point depression and is estimated from an analysis of the fraction of frozen water. Notably, the α values of the polyampholyte cryoprotectant were 8-fold higher than those of the currently used cryoprotectant dimethyl sulfoxide (DMSO) at particular temperatures of interest. Our results illustrate that the presence of a polyampholyte cryoprotectant significantly enhanced the adsorption of the protein/nanocarrier complex onto membranes compared to that obtained with DMSO because of the high freeze concentration. The present study demonstrated the direct relationship between freezing and the penetration of proteins across the periphery of the cell membrane by means of increased concentration during freezing. These results may be useful in providing a guideline for the intracellular delivery of biomacromolecules using ice-crystallization-induced continuous freezing combined with polyampholyte cryoprotectants.

Polyampholytes as Emerging Macromolecular Cryoprotectants

“…This membrane interaction has also been exploited by Matsumura for the introduction of lysozyme proteins into cells through a freeze–thaw mechanism, which enables a 4-fold increase in uptake when compared to nonfrozen cells . It was then demonstrated that these ampholyte-based nanocarrier complexes allowed more protein internalization when compared to 10% DMSO, which is known to enhance membrane permeability . Taken together, these results suggest that while also providing superior cell viability post-thaw, polyampholytes interact with the cell membrane during the freezing process to allow greater payload uptake when incorporated into a protein–nanocarrier complex.…”

Changes in Cryopreserved Cell Nuclei Serve as Indicators of Processes during Freezing and Thawing

“…Cryopreservation of living cells and tissues became fundamental in biotechnology, plant programmes, and modern medicine. − Increasing problems associated with human reproduction put techniques of assisted reproduction, including in vitro fertilization (IVF), to the center of human medicine. In the frame of IVF, embryos and sperm are deeply frozen prior to being mutually coupled, cultured, checked for possible defects, and implanted.…”

Hydrophobic Polyampholytes and Nonfreezing Cold Temperature Stimulate Internalization of Au Nanoparticles to Zwitterionic Liposomes

“…Polyampholytes including zwitterionic polymers also show promise in several other biomedical applications such as for cell preservation, antibiofouling, and tissue engineering. , These polyelectrolytes contain both cationic and anionic charges in the polymeric backbone. We previously developed new hydrophobic polyampholytes that can form self-assembled nanoparticles and efficiently transfer protein-based materials to the cytosol of cells. − These hydrophobic polyampholytes were easily designed by modifying the anionic functional group dodecylsuccinic anhydride (DDSA) and a large amount of succinic anhydride (SA) in ε-poly- l -lysine (PLL), which confers the hydrophobic polyampholytes with an anionic charge. Although hydrophobicity can enhance cell membrane affinity, it is nevertheless difficult to internalize proteins inside cells because of the negatively charged surface potential.…”

“…Although hydrophobicity can enhance cell membrane affinity, it is nevertheless difficult to internalize proteins inside cells because of the negatively charged surface potential. Thus, to achieve efficient delivery to cells, we also developed a novel low temperature-based freeze-concentration approach to act as a driving force to internalize materials inside cells efficiently. − Freeze concentration is a physicochemical phenomenon wherein water molecules are crystallized into ice so that the solutes present in the system are ejected, which thereby enhances the concentration. The increased concentration occurs across the periphery of the cell membrane resulting in adsorption of solutes.…”