(336g) Repurposing Pollen Grains for Oral Delivery of Biologics

In the recent years, use of biologics as therapeutic agents has significantly increased. However, oral delivery of biologics is challenging, especially due to the need to protect them from the harsh environment of the stomach. Other challenges include limited permeability of biologics through the intestinal epithelium. To overcome some of these challenges we have repurposed pollen grains (PGs). PGs are naturally occurring microcapsules with a tough outer shell. Their natural function is to safely transport the male gamete contained within the pollens to the flower for fertilization. To perform this function, the PG wall is naturally tough and can withstand the assault of low pH and enzymes in the stomach. Furthermore, due to microscopic ornamental features on their surface PGs are mucoadhesive, which can increase their residence time over the use of polymeric particles. Thus, we postulated that PGs could be used as ‘Trojan Horses’ to safely carry biologics through the stomach. However, before PGs can be used for oral administration they need to be cleaned to remove intrinsic biomolecules and allergens. Herein, we have developed a new treatment method that uses sequential steps of an organic solvent, acid and an alkali to treat PGs. To augment the properties of PGs we have also created a matrix of an enteric polymer in the PGs to offer protection to the drug and a controlled release property.

Methods: PGs were treated with different protocols to help remove biomolecules from the inner cavity of the PGs, and to obtain clean empty shells. Elemental analysis, scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR) spectroscopy techniques were used to characterize the PGs after treatment. Ragweed (Ambrosia elatior) pollen were then chosen as the model pollen species to evaluate their ability to formulate bovine serum albumin (BSA) as a model biologic. Eudragit L100-55 was used as the enteric polymer and glycofurol was used as the solvent. Formulation was optimized with respect to the amounts of Eudragit L100-55 and BSA that were required to achieve high encapsulation efficiency without causing clumping of PGs (to have them free flowing). The formulation was tested for its ability to protect BSA by incubation in simulated gastric fluid. The release profiles of the formulation were also evaluated in the simulated gastric and intestinal fluids.

Results: SEM and elemental analysis showed that our new chemical treatment method can produce clean PGs from multiple species. FTIR spectra confirmed loss of lipid peaks. Video analysis provided insight into the mechanism of the cleaning steps. After cleaning, apertures, which are naturally weak spots in the PG wall got opened producing orifices in the pollen wall measuring about 1 μm in diameter. To ensure that encapsulated proteins do not leak out from these orifices we successfully produced a matrix of enteric Eudragit L100-55 in the PGs. BSA was encapsulated at about 12% efficiency in this matrix. When PGs with Eudragit+BSA were incubated in simulated gastric fluid, the matrix offered protection against denaturation as determined through measurement of intrinsic fluorescence of tyrosine and tryptophan residues. Lastly, we measured the release kinetics of the PGs with Eudragit+BSA in simulated gastric and intestinal fluids. As expected, the presence of PGs provided controlled release, and this effect was accentuated with the use of Eudragit L100-55. The formulation had a slower release as compared to just the Eudragit polymer. We also confirmed the mucoadhesive nature of ragweed PGs by their ability to stick to ex vivo pig intestine, offering PGs a significant edge over smooth polymeric particles.

Conclusions and comparison of PGs to synthetic polymeric particles: These results demonstrate the ability of our new treatment protocol to clean a variety of PGs, and pave the way to investigate various applications of these naturally-occurring pollen particles. Furthermore, the protection of proteins and peptides in the PGs using the enteric matrix creates a new oral delivery system that could substitute parenterally administered biologics and vaccines. The natural mucoadhesive property of PGs can significantly increase their residence time, which makes them very attractive over synthetic polymeric particles. Synthetic polymeric particles also often require the use of organic solvents for encapsulation, and this can harm the proteins to be encapsulated. In contrast glycofurol is protein-friendly. From a cost and manufacturing perspective, PGs are already commercially harvested in large amounts to cater to the need of the allergy industry. In allergen field, PGs are incubated with buffers to extract allergens for immunotherapy and allergy testing. The PG shells are a waste product of this industry. Cost wise PGs are relatively inexpensive. Overall, use of PGs for oral delivery of biologics is an interesting and a viable option that should receive more attention.