Sana Ahmed

Protein cytoplasmic delivery using polyampholyte nanoparticles and freeze concentration

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.

A Freeze-Concentration and Polyampholyte-Modified Liposome-Based Antigen-Delivery System for Effective Immunotherapy

&NA; Immunotherapy is an exciting new approach to cancer treatment. The development of a novel freeze‐concentration method is described that could be applicable in immunotherapy. The method involves freezing cells in the presence of pH‐sensitive, polyampholyte‐modified liposomes with encapsulated ovalbumin (OVA) as the antigen. In RAW 264.7 cells, compared to unfrozen, freeze‐concentration of polyampholyte‐modified liposomes encapsulating OVA resulted in efficient OVA uptake and also allowed its delivery to the cytosol. Efficient delivery of OVA to the cytosol was shown to be partly due to the pH‐dependence of the polyampholyte‐modified liposomes. Cytosolic OVA delivery also resulted in significant up‐regulation of the major histocompatibility complex class I pathway through cross‐stimulation, as well as an increase in the release of IL‐1β, IL‐6, and TNF‐α. The results demonstrate that the combination of a simple freeze‐concentration method and polyampholyte‐modified liposomes might be useful in future immunotherapy applications.

pH-Responsive Polyion Complex Vesicle with Polyphosphobetaine Shells

When a bioactive molecule is taken into cells by endocytosis, it is sometimes unable to escape from the lysosomes, resulting in inefficient drug release. We prepared pH-responsive polyion complex (PIC) vesicles that collapse under acidic conditions such as those inside a lysosome. Furthermore, under acidic conditions, cationic polymer was released from the PIC vesicles to break the lysosome membranes. Diblock copolymers (P20M167 and P20A190) consisting of water-soluble zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) block and cationic or anionic blocks were synthesized via reversible addition-fragmentation chain transfer (RAFT) radical polymerization. Poly(3-(methacrylamidopropyl) trimethylammonium chloride) (PMAPTAC) and poly(sodium 6-acrylamidohexanoate) (PAaH) were used as the cationic and anionic blocks, respectively. The pendant hexanoate groups in the PAaH block are ionized in basic water and in phosphate buffered saline (PBS), while the hexanoate groups are protonated in acidic water. In basic water, PIC vesicles were formed from a charge neutralized mixture of oppositely charged diblock copolymers. At the interface of PIC vesicle and water exists biocompatible PMPC shells. Under acidic conditions, the PIC vesicles collapsed, because the charge balance shifted due to protonation of the PAaH block. After collapse of the PIC vesicles, P20A190 formed micelles composed of protonated PAaH core and PMPC shells, while P20M167 was released as unimers. PIC vesicles can encapsulate hydrophilic nonionic guest molecules into their hollow core. Under acidic conditions, the PIC vesicles can release the guest molecules and P20M167. The cationic P20M167 can break the lysosome membrane to efficiently release the guest molecules from the lysosomes to the cytoplasm.

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

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.

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

Nanomedicine relies on the effective internalization of nanoparticles combined with polymeric nanocarriers into living cells. Thus, exploration of internalization is essential for improving the efficacy of nanoparticle-based strategies in clinical practice. Here, we investigated the physicochemical internalization of gold nanoparticles (AuNPs) conjugated with hydrophobic polyampholytes into cell-sized liposomes at a low but nonfrozen temperature. The hydrophobic polyampholytes localized in the disordered phase of the membrane, and internalization of AuNPs was enhanced in the presence of hydrophobic polyampholytes together with incubation at -3 °C as compared to 25 °C. These results contribute toward a mechanistic understanding for developing a model nanomaterials-driven delivery system based on hydrophobic polyampholytes and low temperature.