Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells

Abstract

Targeted oral delivery of GFP fused with a GM1 receptor binding protein (CTB) or human cell penetrating peptide (PTD) or dendritic cell peptide (DCpep) was investigated. Presence of GFP+ intact plant cells between villi of ileum confirm their protection in the digestive system from acids/enzymes. Efficient delivery of GFP to gut-epithelial cells by PTD or CTB and to M cells by all these fusion tags confirm uptake of GFP in the small intestine. PTD fusion delivered GFP more efficiently to most tissues or organs than other two tags. GFP was efficiently delivered to the liver by all fusion tags, likely through the gut-liver axis. In confocal imaging studies of human cell lines using purified GFP fused with different tags, GFP signal of DCpep-GFP was only detected within dendritic cells. PTD-GFP was only detected within kidney or pancreatic cells but not in immune modulatory cells (macrophages, dendritic, T, B, or mast cells). In contrast, CTB-GFP was detected in all tested cell types, confirming ubiquitous presence of GM1 receptors. Such low-cost oral delivery of protein drugs to sera, immune system or non-immune cells should dramatically lower their cost by elimination of prohibitively expensive fermentation, protein purification cold storage/transportation and increase patient compliance.

Expression and functional evaluation of biopharmaceuticals made in plant chloroplasts

“…Delivery of CTB fusion proteins across the blood-brain barrier or blood-retinal barrier was also observed in recent studies performed in mice [9••,10••]. Once delivered to the small intestine, proteins are taken up by the gut epithelial or microfold cells (M cells) before they enter the circulation system or protein drugs enter via the gut-liver axis [7••]. CTB fusion protein was detected in both human non-immune and immune modulatory cells [7••].…”

“…When fused with suitable tags, protein drugs cross gut epithelial cells and enter into the circulatory or immune system [5–8]. Recent studies show that oral delivery of protein to different cell types can be achieved using different transmucosal peptides fused to the N- or C-terminus of recombinant proteins [7••, Figure 1]. When cholera non-toxic B subunit (CNTB), protein transduction domain (PTD) or dendritic cell peptide (DCpep) were fused with GFP, fluorescence was widely distributed in mouse respiratory, digestive and skeletal muscle or other tissues or organs.…”

“…Once delivered to the small intestine, proteins are taken up by the gut epithelial or microfold cells (M cells) before they enter the circulation system or protein drugs enter via the gut-liver axis [7••]. CTB fusion protein was detected in both human non-immune and immune modulatory cells [7••]. Recent reports also show that antimicrobial peptide (AMP), Protegrin-1 (PG-1) enters various human cell types including periodontal ligament stem cells, head and neck squamous cell carcinoma cells, Gingiva-derived mesenchymal stromal cells and adult gingival keratinocytes more efficiently than any other known cell penetrating peptides [11••].…”

“…(c) Biodistribution of GFP signal in different tissues of mice fed with lyophilized leaves expressing GFP fused with PTD, CTB and Dcpep tags. The figure was modified from previous publication from our lab [7••, 8]. …”

Topical delivery of low-cost protein drug candidates made in chloroplasts for biofilm disruption and uptake by oral epithelial cells

“…The transplastomic plants expressing green fluorescence protein (GFP) fused with Cholera Toxin B subunit (CTB), Protein Transduction Domain (PTD), retrocyclin and protegrin were created as described in previous studies [16,38,44,45]. Purification of GFP-fused PG1 or RC101 from transplastomic tobacco was done from 0.2–1 gm of lyophilized plant material.…”