Efficient intracellular delivery of p53 protein by engineered protein crystals restores tumor suppressing function in vivo.

Direct delivery of proteins into cells holds significant potential for basic research and drug development. However, the poor endosomal escape of conventional delivery strategies remains a challenge, thus limiting the clinical translation of many protein therapeutics. Herein, we report that engineered Cry3Aa protein (Pos3Aa) crystals formed naturally within Bacillus thuringiensis can serve as a vehicle for efficient cytosolic delivery of bioactive proteins. We showed that Pos3Aa-mediated delivery of tumor suppressor p53 protein, a promising therapeutic candidate found to be inactivated in nearly half of human cancers, resulted in the restoration of p53 function in p53-deficient cancer cells, and thereby sensitized them to 5-fluorouracil chemotherapy as demonstrated in in vitro and in vivo models. Our results validate that Pos3Aa crystals can be a robust and effective platform for the cytosolic delivery of effector proteins, and suggest that efficient uptake and endosomal escape could be critical for efficacious p53 protein-based cancer therapy.

Targeted Myoglobin Delivery as a Strategy for Enhancing the Sensitivity of Hypoxic Cancer Cells to Radiation.

The effectiveness of cancer radiotherapy is frequently hindered by the hypoxia of the tumor microenvironment. Direct delivery of oxygen to hypoxic tumor tissues is an attractive strategy to overcome this hypoxia-associated radioresistance. Herein, we report the generation of submicron-sized particles comprising myoglobin fused to the crystal-forming domain of Cry3Aa protein for the targeted delivery of oxygen to cancer cells. We demonstrate that myoglobin-containing particles were successfully produced in Bacillus thuringiensis with the assistance of the Cry3Aa domain I. Furthermore, these particles could be genetically modified to incorporate the cell penetrating peptide TAT and cell targeting peptide A549.1, resulting in particles that exhibited improved cellular uptake and targeting toward A549 cells. Notably, these myoglobin-containing particles increased the intracellular oxygen levels of A549 cells and thereby sensitized them to radiation. These findings suggest that the targeted delivery of O2-bound myoglobin could be an effective approach to enhance the efficacy of radiotherapy.

Targeted delivery of antimicrobial peptide by Cry protein crystal to treat intramacrophage infection.

Antimicrobial peptides (AMPs) have recently attracted great attention due to their rapid action, broad spectrum of activity, and low propensity of resistance development. The successful application of AMPs in the treatment of intracellular infections, however, remains a challenge because of their low penetration efficiency into the pathogen's intracellular niche. Herein, we report that sub-micrometer-sized crystals of the protein Cry3Aa formed within Bacillus thuringiensis are readily and specifically taken up by macrophages. We demonstrate that these protein crystals efficiently encapsulate a known antileishmanial peptide, dermaseptin S1 (DS1), and thereby promote improved cellular uptake of DS1 and its lysosomal accumulation in macrophages. Notably, this targeted delivery of DS1 results in enhanced in vitro and in vivo antileishmanial activity, as well as reduced toxicity to the host macrophages. These findings suggest that the Cry3Aa crystal can be an effective delivery platform for AMPs to treat intramacrophage infections.