(A-3) Bacterial cellulose nanoparticles as a scalable, sustainable drug delivery platform

Introduction:: The nanomedicine market is a rising sector where a major focus is scaled-up manufacturing. Recent examples such as the SARS-coronavirus-19 vaccine showed critical challenges in manufacturing including large scale and waste-free production, cold chain shipping, and shelf-life stability. Thermostability of the vaccine was the most apparent burden – cryogenic temperatures at -80°C and refrigeration between +2°C and +8°C were crucial to ensure sample stability and shelf-life.[1] Manufacturing techniques such as Particle Replication In Non-wetting Templates (PRINT) address scalability and reproducibility but are still challenged by storage and waste production,[2] where solvent disposal creates environmental challenges.[3] Therefore, it becomes critical to create sustainable manufacturing processes for nanotherapeutics to reduce environmental impact and increase the cost effectiveness in manufacturing. Cellulose produced by bacterial species (bacterial cellulose, BC) is an emerging biopolymer platform used in engineering and biomedical applications. In an aqueous and low-cost media enriched with microbial co-cultures, known as a kombucha media, some bacterial species produce pure cellulose.[4] BC is a versatile biopolymer platform and can address the challenge of sustainable manufacturing and commercialization of therapeutics. BC microparticles have been previously produced in agitated and aerated conditions,[4-8] but are limited for deep tissue penetration by their size. A nanoparticle-based BC can provide longer circulation times, improved bioavailability and biodistribution, and greater targeted delivery through increased tissue penetration and cell specific uptake. Here we report the development of BC nanoparticles (BCNPs) as a drug delivery platform that can address the challenges of sustainability, scalability, reproducibility, and thermal stability.

Materials and Methods:: BCNPs were prepared in agitated and aerated conditions in a kombucha media on an orbital plate shaker at 150RPM and 30°C for 24h. The tea media was prepared with a nitrogen sourced black tea steeped in hot deionized (DI) water, white pure cane sugar, and apple cider vinegar. Suspensions extracted from a Symbiotic Culture of Bacteria and Yeast (SCOBY) starter were extracted to inoculate the tea media. At 24h, 0.1M sodium hydroxide solution was titrated into the kombucha media to a slight excess to terminate the growth of cellulose.[4, 9] The BCNPs were harvested from media with vacuum filtration and cleaned from excess media and NaOH via dialysis tubing made of cellulose ester. A 100K centrifugal tube filter centrifuged at 4000RPM separated BCNPs from solution. To size separate further, BCNPs suspended in water were bath sonicated with 0.1M polysorbate 80 surfactant solution and size filtered with a 200nm syringe filter. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), nanoparticle tracking analysis, dynamic light scattering and scanning transmission electron microscopy were the characterization techniques used for BCNPs. BCNPs were then incorporated with bovine serum albumin (BSA), serving as the model drug, and incubated for 2h at ambient temperature on an orbital rotator mixer, collected, and washed in DI twice at 10,000 × g centrifugation. The supernatant was collected from each wash and combined for BSA quantification using commercial bicinchoninic acid (BCA) protein assay kit and ultra-violet spectroscopy.


Results, Conclusions, and Discussions:: BCNPs are an innovative platform in drug delivery; BC is scalable and sustainable, and produces modifiable particle size, morphology, surface charge, while allowing a tunable degree of crystallinity from purely amorphous to highly crystalline, all of which are desired attributes in biomedical applications.[4, 10, 11] Tuning the physiochemical properties of BCNPs is necessary for developing a therapeutic platform that can selectively localize at target disease sites. In this study, BCNPs were prepared with P80 surfactant solution and size separated to 103.0 ± 1.5nm with a near neutral zeta-potential of -2.3 ± 2.0mV. Agitation enabled the cellulose nanofibers to take a spherical shape, making BCNPs a viable option for a drug delivery system in tissue penetration and cell specific uptake applications. [4, 9] XRD, FTIR, and TGA characterized the physiochemical properties of BCNPs for crystallinity, chemical bonding, and thermal degradation behavior analysis. XRD showed an amorphous material structure for BCNPs grown for 1 day. We observed that longer culture times result in increased crystallinity. Aeration and agitation are other noted factors that affect the crystallinity and degree of polymerization of BC.[4, 12] The characteristic peaks of BC were identified and amongst those, the broadened (-OH) peaks at 3200-3400 cm^-1 show the dominant hydrogen bonding features and the (C-O-C) peak at 1163 cm^-1 show the β (1-4) glycosidic linkages. [6, 13] TGA confirmed the stability of the BC samples above biological temperatures (degradation starts at ~150°C) that will significantly impact shipping and storage conditions. BSA incorporated into BCNPs demonstrated high loading efficiency percentages with approximately 5%, 10%, and 20% drug loading. BSA was chosen as a therapeutic because of its strong binding affinity and electrostatic interactions with cellulose. [4] BSA is also low cost with a high aqueous solubility and can be incorporated into BC as a protein therapeutic.[4, 14, 15] Our results show that BCNPs are a promising alternative drug delivery therapeutic that provide reduced environmental impact through use of green chemistry and green methodologies. BCNPs will provide an avenue for a future of sustainable drug delivery technologies.


Acknowledgements (Optional): : The authors thank Jeremy Filteau for assisting with BCA assay training and Ellen Lavoie for assisting with STEM sample preparations and instrument training at the MAF.


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