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 degrees 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 degrees 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 (alpha) that depends on the freezing-point depression and is estimated from an analysis of the fraction of frozen water. Notably, the a 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.