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. 2016 Oct 24:6:35765.
doi: 10.1038/srep35765.

Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs

María Virtudes Céspedes  1   2 , Yolanda Fernández  2   3 , Ugutz Unzueta  1   2 , Rosa Mendoza  2   4 , Joaquin Seras-Franzoso  2   3 , Alejando Sánchez-Chardi  5 , Patricia Álamo  1   2 , Verónica Toledo-Rubio  2   4   6 , Neus Ferrer-Miralles  2   4   6 , Esther Vázquez  1   2 , Simó Schwartz  2   3 , Ibane Abasolo  2   3 , José Luis Corchero  2   4   6 , Ramon Mangues  1   2 , Antonio Villaverde  2   4   6
Affiliations

Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs

María Virtudes Céspedes et al. Sci Rep. .

Abstract

In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4+ cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals' endocrine system.

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Figures

Figure 1
Figure 1. Morphometric analysis of amyloid materials at structural and ultrastructural levels.
(A) Representative FESEM overviews and details (inboxes) of the amyloidal particulate materials used for immobilization, which occurred as rather homogenous populations. Size range of IBVP1TFP was 400–500 nm, 200–300 nm for IBVP1GFP and 500–600 nm for IBT22-GFP-H6 (n > 200). Size bars are common in all images and all inboxes as magnifications are equivalent. (B) Broad field confocal images of the materials and 3D IMARIS reconstructions of representative particles (inboxes). Particle sizes here might be largely overestimated due to fluorescence emission.
Figure 2
Figure 2. Whole-body biodistribution of fluorescent amyloids upon intravenous or intratumoral administration in a HT-29 colorectal cancer model.
(A) Ex vivo lung fluorescence imaging (FLI) at 4 and 24 h post administration of IBVP1TFP protein particles at 60 μg/mouse by intravenous administration route. Ex vivo lung-accumulation of the material was quantified by measuring fluorescent intensity (left plot), while representative ex vivo fluorescent images of the excised lungs are shown (right panel). (B) Non-invasive monitoring of IBVP1TFP tumor-accumulations a long time after intratumoral administration at 12, 24 and 60 μg/mouse of IBVP1TFP. In vivo tumor-accumulation of the material was quantified (left plot), and representative in vivo fluorescent image of tumor-accumulation at 24 h post administration are shown (right panel). (C) Ex vivo tumor FLI at 4 h after intratumoral administration at 12, 24 and or 60 μg/mouse, and 7 days after intratumoral administration of 12 μg/mouse of the material. Ex vivo tumor-accumulation of IBVP1TFP was quantified (left plot), and representative ex vivo fluorescent images of the excised tumor (whole- and sectioned-tumor) are shown (right panel). In all cases, the total tissue-accumulations were quantified by measurements of fluorescent intensity expressed in Radiant Efficiency, MEAN ± SEM. Pseudocolor scale bars were consistent for all images in order to show relative changes for each corresponding images.
Figure 3
Figure 3. Whole-body biodistribution of functional fluorescent amyloids in CXCR4+ colorectal murine model.
(A) Representative ex vivo fluorescent images of the excised tumor and organs (brain, liver, kidney and lung and heart) remaining in tumor tissue at 5 h, and 3 or 7 days post administration of IBT22-GFP-H6 protein particles at 60 μg/mouse or 200 μg/mouse using the intratumoral route. (B) Fluorescence emitted by soluble protein species in plasma, which were released by IBT22-GFP-H6 tumor deposits to the bloodstream at 5 h, and 3 or 7 days after injection of a 200 μg/mouse intratumoral dose. Pseudocolor scale bars were consistent among images in order to show relative changes when being compared.
Figure 4
Figure 4. Fluorescent emission by IBT22-GFP-H6 deposits remaining in CXCR4+ colorectal tumors after their administration.
(A) Total IBT22-GFP-H6 protein deposits were quantified measuring fluorescent intensity at 5h, 3 and 7 days after intratumoral injection of 200 μg/mouse. Data were expressed in Radiant Efficiency. (B) Quantitation of total IBT22-GFP-H6 protein deposits plus released soluble proteins in tumor tissue calculated as an H-score for anti-GFP immunostaining (brown colour) at 5 h, and 3 or 7 days post administration. (C) Representative microphotographs of GFP inmunohistochemistry in IBT22-GFP-H6 treated tumors at 5 h, and 3 or 7 days. Note the higher intensity of GFP staining in some tumor areas at 5 h and 3 days (black arrows) and the higher dispersion of protein distribution observed inside the tumor at day 7 post-injection. Quantitative data were expressed as mean ± SE *,**Statistically significant at p < 0.05 or p < 0.01, respectively.
Figure 5
Figure 5. Apoptotic and mitotic index recorded in CXCR4+ tumors.
Quantitation of apoptotic figures detected by nuclear condensation or nuclear fragmentation after Hoescht staining (A) or mitotic figures (B) after Hematoxylin-eosin staining in SP5 tumors at day 7 post intratumoral administration of IBT22-GFP-H6 protein particles at 60 or 200 μg/mouse. (C,D) Representative microphotographs of apoptotic figures by Hoescht ((C), white arrows) or HE ((D), white arrows) staining or mitotic figures ((D), black arrows) by HE staining after local intratumoral injection of 200 μg/mouse dose of targeted IBT22-GFP-H6 or non-targeted IBVP1GFP (x 400 magnification). (E) Quantitation of the number of positive cells displaying cleaved (active) caspase-3 immunostaining in tumors at 5 h, and 3 or 7 days after intratumoral administration of 200 IBT22-GFP-H6 compared to non-treated tumors. (F) Representative microphotographs of active caspase-3 positive cells (brown stained tumor cells; black arrows) at the studied time point. Data expressed as mean ± SE *,**Statistically significant at p < 0.05 or p < 0.01, respectively.
Figure 6
Figure 6. Protein release from IBs.
(A) Immunogold labeling of GFP in HeLa cells after 24 h of exposure to VP1-GFP-H6 IBs. Blue arrows indicate labeling in the IB particle while red arrows indicates labeling of released protein to the cell cytoplasm. Highly electrodense IB particles with standard morphometries are shadowed in red while those with loose morphologies and showing lower electrodensity, in yellow. The scale bar represents 200 nm. (B) Quantification of IB-attached and IB-free immunolabeling signals at this incubation time.
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