Cry Protein Crystals: A Novel Platform for Protein Delivery

<div><p>Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium <i>Bacillus thuringiensis</i>. We demonstrate that fusion of the <i>cry3Aa </i>gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines <i>in vitro</i>, and can be delivered to mice <i>in vivo</i> via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.</p></div

Fluorescence and TEM images of Cry3Aa-GFP and Cry3Aa-mCherry crystals uptaken into cells.

<p>Macrophage cells (RAW264.7) were incubated with Cry3Aa-GFP crystals for different time periods. Fluorescence images at (a) 15-min, demonstrating fast initial uptake and at (b) 1-h, where the green fluorescence intensity reached its maximum value. Note the punctate fluorescence pattern, which supports uptake of the whole crystal. (c) Red fluorescence image of primary mouse fibroblasts after 1.5 h incubation with Cry3Aa-mCherry protein crystals is not as intense as seen with macrophages. TEM images of (d) untreated RAW264.7 cells, and (e) cells treated with Cry3Aa-GFP. The Cry3Aa-GFP treated cells exhibit a distinct cytoplasmic particulate not observed in the control cells. (f) Enlarged TEM image of the cytoplasmic particulate observed in the Cry3Aa-GFP treated macrophages highlighting the likely crystal degradation in the cytoplasm.</p

Bioluminescence of Cry3Aa-luciferase crystals delivered to mice.

<p>(a) No Cry3Aa-luciferase crystal control (b) nasal uptake, and (c) intraperitoneal injection. The luminescence measurements were taken 10 min after crystal and D-luciferin delivery. (d) Luciferase activity following oral gavage of Cry3Aa-luciferase crystals after 5, 10, 20, 30, 40, and 50 min. The D-luciferin substrate was replenished at 20 min intervals. Activity and lifetime of luciferase activity of Cry3Aa-luciferase crystals (0.5 mg or 0.25 mg as indicated in the figure) and luciferase protein (2.5 mg or 1.25 mg as indicated in the figure) delivered to C57BL/6 mice via (e) nasal spray, (f) intraperitoneal injection and (g) oral gavage. Measurements were made by selecting a region of interest (ROI) in the area where the maximal intensity was obtained from the crystal and the protein. Lifetime of luminescence was measured in the selected ROI over the indicated times. D-luciferin was replenished every 20 min. The lifetime of the luciferase activity of the Cry3Aa-luciferase crystals was found to be higher that of the recombinant luciferase protein in each route of delivery, supporting the ability of the Cry3Aa crystal framework to protect the luciferase protein from degradation.</p

Uptake of Cry3Aa-GFP crystals by antigen presenting cells in Peyer’s patches.

<p>(a) Percentage of IAb⁺ cells (MHC-class II) in Peyer’s patches that retained GFP after 8 hours post oral intubation of Cry3Aa-GFP crystals or GFP protein (GFP⁺IAb⁺). (b) Percentage of CD11b⁺ cells (macrophages) in Peyer’s patches that retained GFP after 8 hours post oral intubation of Cry3Aa-GFP crystals or GFP protein (GFP⁺CD11b⁺). Data are represented as mean ± std. dev of samples assayed; * (<i>P<0</i>.<i>05</i>); ** (<i>P<0</i>.<i>01</i>). The flow cytometry analyis of GFP⁺ live cells was made with an Accuri C6 flow cytometer after immunostaining of cell surface markers with fluorescent antibodies.</p