Cytosolic protein delivery is of central importance for the development of protein-based biotechnologies and therapeutics; however, efficient intracellular delivery of native proteins remains a challenge. Here, we reported a boronic acid–rich dendrimer with unprecedented efficiency for cytosolic delivery of native proteins. The dendrimer could bind with both negatively and positively charged proteins and efficiently delivered 13 cargo proteins into the cytosol of living cells. All the delivered proteins kept their bioactivities after cytosolic delivery. The dendrimer ensures efficient intracellular delivery of Cas9 protein into various cell lines and showed high efficiency in CRISPR-Cas9 genome editing. The rationally designed boronic acid–rich dendrimer permits the development of an efficient platform with high generality for the delivery of native proteins.

We herein report an optogenetically activatable CRISPR-Cas9 nanosystem for programmable genome editing in the second near-infrared (NIR-II) optical window. The nanosystem, termed nanoCRISPR, is composed of a cationic polymer-coated Au nanorod (APC) and Cas9 plasmid driven by a heat-inducible promoter. The APC not only serves as a carrier for intracellular plasmid delivery but also can harvest external NIR-II photonic energy and convert it into local heat to induce the gene expression of the Cas9 endonuclease. Due to high transfection activity, the APC shows strong ability to induce a significant level of disruption in different genomic loci upon optogenetic activation. Moreover, the precise control of genome-editing activity can be simply programmed by finely tuning exposure time and irradiation time in vitro and in vivo and also enables editing at multiple time points, thus proving the sensitivity and inducibility of such an editing modality. The NIR-II optical feature of nanoCRISPR enables therapeutic genome editing at deep tissue, by which treatment of deep tumor and rescue of fulminant hepatic failure are demonstrated as proof-of-concept therapeutic examples. Importantly, this modality of optogenetic genome editing can significantly minimize the off-target effect of CRISPR-Cas9 in most potential off-target sites. The optogenetically activatable CRISPR-Cas9 nanosystem we have developed offers a useful tool to expand the current applications of CRISPR-Cas9, and also defines a programmable genome-editing strategy toward high precision and spatial specificity.

Purpose: Tumor-derived exosomes are proposed as a new type of cancer vaccine. Heat shock proteins are potentTh1adjuvant, and heat stress can induce heat shock protein and MHC-I expression in tumor cells, leading to the increased immunogenicity of tumor cells. To improve the immunogenicity of exosomes as cancer vaccine, we prepared exosomes from heat-stressed carcinoembryonic antigen (CEA)^positive tumor cells (CEA +

A major challenge in vaccine formulations is the stimulation of both the humoral and cellular immune response for well-defined antigens with high efficacy and safety. Adjuvant research has focused on developing particulate carriers to model the sizes, shapes and compositions of microbes or diseased cells, but not antigen fluidity and pliability. Here, we develop Pickering emulsions-that is, particle-stabilized emulsions that retain the force-dependent deformability and lateral mobility of presented antigens while displaying high biosafety and antigen-loading capabilities. Compared with solid particles and conventional surfactant-stabilized emulsions, the optimized Pickering emulsions enhance the recruitment, antigen uptake and activation of antigen-presenting cells, potently stimulating both humoral and cellular adaptive responses, and thus increasing the survival of mice upon lethal challenge. The pliability and lateral mobility of antigen-loaded Pickering emulsions may provide a facile, effective, safe and broadly applicable strategy to enhance adaptive immunity against infections and diseases.

The phosphatidylethanolamine (PE)-binding proteins (PEBPs) are an evolutionarily conserved family of proteins with pivotal biological functions. Here we describe the cloning and functional characterization of a novel family member, human phosphatidylethanolaminebinding protein 4 (hPEBP4). hPEBP4 is expressed in most human tissues and highly expressed in tumor cells. Its expression in tumor cells is further enhanced upon tumor necrosis factor (TNF) ␣ treatment, whereas hPEBP4 normally co-localizes with lysosomes, TNF␣ stimulation triggers its transfer to the cell membrane, where it binds to Raf-1 and MEK1. L929 cells overexpressing hPEBP4 are resistant to both TNF␣-induced ERK1/2, MEK1, and JNK activation and TNF␣-mediated apoptosis. Co-precipitation and in vitro protein binding assay demonstrated that hPEBP4 interacts with Raf-1 and MEK1. A truncated form of hPEBP4, lacking the PE-binding domain, maintains lysosomal co-localization but has no effect on cellular responses to TNF␣. Given that MCF-7 breast cancer cells expressed hPEBP4 at a high level, small interfering RNA was used to silence the expression of hPEBP4. We demonstrated that down-regulation of hPEBP4 expression sensitizes MCF-7 breast cancer cells to TNF␣-induced apoptosis. hPEBP4 appears to promote cellular resistance to TNF-induced apoptosis by inhibiting activation of the Raf-1/MEK/ERK pathway, JNK, and PE externalization, and the conserved region of PE-binding domain appears to play a vital role in this biological activity of hPEBP4.The phosphatidylethanolamine-binding protein (PEBP) 1 family consists of a number of 21-23-kDa basic proteins, first identified in bovine brain, with preferential in vitro affinity for phosphatidylethanolamine, a component of the cell membrane. This family is an evolutionarily conserved group found in species of flowering plants (Antirrhinum (1)), parasites (Plasmodium falciparium (2)), nematodes (Toxocara canis (3)), insects (Drosophila melanogaster (4)), and mammals, including cattle, monkeys, and humans (5). A number of functions have been suggested for the mammalian PEBP proteins, including lipid binding and inhibition of serine proteases (6). These proteins can also act as precursors for a bioactive peptide HCNP (hippocampal cholinergic neurostimulating peptide), important in hippocampus development (5). Plant PEBP homologues are involved in the control of a morphogenic switch between shoot growth and flower structures (7). Yeast two-hybrid screen analysis has shown that human PEBP1 (hPEBP1, also called Raf kinase inhibitory protein or RKIP) acts as a suppressor of Raf-1 kinase activity and mitogen-activated protein kinase signaling in fibroblasts via its ability to sequester and inactivate Raf-1 and MEK1 (8, 9). Both Raf-1 and MEK bind to the highly conserved phosphatidylethanolamine-binding domain of hPEBP; hPEBP induces dissociation of Raf-1⅐MEK complexes and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains has shown that MEK and Raf-1 bind to overlapping sites in hPEBP...