Genetically engineered Spirulina plantensis producing human insulin as a potential novel oral drug delivery carrier

Abstract

Oral drug delivery has always been the preferred route for patients and physicians alike. However, the oral route presents several challenges, including low permeability and uptake, particularly for therapeutic proteins, with insulin being a prime example. Oral insulin barely reaches the bloodstream as it is digested and breaks down due to the enzymatic environment and its inability to transverse the gut barrier due to its molecular weight. To the best of our knowledge, this is the first report to investigate the potential of genetically engineered (GE) Arthrospira platensis (i.e., Spirulina) as an oral carrier of human insulin (hINS), as well as the intestinal uptake of recombinant hINS produced by GE Spirulina. Furthermore, we report for the first time the application of acetosyringone (ACE) and salicylic acid (SA) to GE Spirulina to enhance hINS production. This study examines the potential of GE Spirulina as an oral delivery vehicle. Agrobacterium containing a pCAMBIA-based binary vector with the hINS gene in its transfer-DNA (T-DNA) segment was used to transform Spirulina via the co-cultivation method. Acetosyringone (ACE) was investigated for its effect on transformation efficiency, and salicylic acid (SA) was investigated for its ability to increase hINS production. The production of hINS was quantified using the hINS ELISA kit. Furthermore, we investigated the scalability potential of this method using laboratory-scale batch bioreactors. Lastly, the potential of engineered Spirulina to deliver hINS was assessed via intestinal uptake and permeability experiments using rat tissues. Colony-PCR confirmed the presence of pCAMBIA-based plasmid in Agrobacterium tumefaciens GV3101 (used to transform Spirulina). Spirulina was found to be sensitive to hygromycin and cefotaxime antibiotics, enabling the harvesting and separation of transformed Spirulina. The PCR amplification of hINS from genomic DNA indicated stable integration into the Spirulina genome, consistent with previous reports; however, additional junction-level confirmation is recommended. Adding 200 µM ACE during the transformation process increased transfection and hINS production compared to the control (2.3 ± 0.15 ng vs. 0.9 ± 0.31 ng, respectively). A second round of transformation increased hINS production while a third transfection cycle reduced it. The addition of 100 µM SA increased hINS production by 2.9-fold. Interestingly, scaled-up production of genetically engineered (GE) Spirulina using a 2 L glass bioreactor resulted in 277% higher hINS production than expected based on volume increase. The hINS produced by GE Spirulina is bioactive, is released intact, and remains stable under intestinal conditions. Ex vivo experiments with the small intestines of rats exposed to GE Spirulina revealed that hINS was uptaken in the rat's ileum > jejunum > duodenum. In contrast, transverse accumulation of the hINS was the highest for the duodenum, followed by the jejunum and ileum. The results of this study demonstrate the potential of GE Spirulina as an effective oral drug carrier, warranting further exploration.