(163g) Protein-Loaded, Sustainably Formulated Bacterial Cellulose Nanoparticles

Background: Many U.S. FDA approved materials in nanomedicine technologies involve chemically intensive syntheses and are difficult to manufacturer at the kilogram scale. Our prior work used copolymer poly(lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG) to deliver drugs to the injured newborn brain. Although PLGA-PEG is a viable and effective platform, the synthetic material is limited by scalability and sustainability. Green engineering is one way to address the concerns of sustainability in formulation of nano-based delivery systems. Here, we develop bacterial cellulose nanoparticles (BCNPs) for sustainable drug delivery applications and motivated by the material’s bio renewable sourcing and biocompatibility, which are important properties for nanomedicine applications. BCNPs are a therapeutically relevant formulation that has a reduced environmental impact, an overall eco-friendly life cycle (Figure 1) and can be implemented following greener engineering methodologies.

Methods: We applied surface functionalizations to BC to tune the surface functionality and charge density. To achieve the modifications, we utilize literature reported strategies, such as methylation, acetylation, and amination. We first used commercially manufactured alpha cellulose and surface modified via methylation and acetylation to yield a greater hydrophobic material, while amination was used to yield a more hydrophilic material. After validating the functionalized alpha cellulose for positive and negatively charged cellulose matrices with Fourier transform infrared spectroscopy (FTIR) and X-ray crystallography (XRD), we next applied the same functionalization methods to BC pellicle. The BC pellicle was first collected from a kombucha co-culture media (bacteria and yeast) from the air and water interface, washed with sodium hydroxide and water for isolation and purifications steps, and then dissolved in small volumes of dimethylacetamide and lithium chloride. The BC pellicle was then functionalized using the same methods applied to alpha cellulose. After functional groups were confirmed, the BC pellicle was processed to create a small library of functionalized BCNPs for protein loading applications. The dissolved BC pellicle underwent a double emulsion formulation with polysorbate 80 surfactant solution to produce BCNPs. To make sure the functional groups were retained when making BCNPs from functionalized BC pellicle, we again performed FTIR and XRD. Slightly changing the pH during or post modification of the BCNPs allowed greater particle dispersion upon drying and redispersion in water. The particle size, zeta-potential, and polydispersity index (PDI) were measured by dynamic light scattering, NanoSizer, and electron microscopy.

Cytotoxicity of BCNPs were evaluated in our established organotypic whole hemisphere (OWH) brain slice platform prepared from postnatal day 10 (P10) rat brains and compared to chitosan nanoparticles and alginate nanoparticles, field standards for polysaccharide-based nanoparticles. After plating 300 µm OWH slices on semi-permeable membranes in slice culture media, slices were cultured for 4 days in vitro (DIV). At 4 DIV, BCNPs were topically applied to slices at 3 doses: 5 µg, 25 µg, 75 µg. After 24 h of BCNPs exposure, slices were stained with propidium iodine (PI), a cell death stain, and TO-PRO-3, a positive nuclei stain. Slices were fixed in methanol and PI+ cells were imaged using confocal microscopy. A separate set of slices exposed to BCNPs were collected and RNA was isolated to perform qPCR analysis to measure cytokine response. Cellular activation markers that were assessed include: Ki67, CD68, GFAP, Synapsin, CD11b, Vim. Lastly, to demonstrate the ability to load catalase into BCNPs, we incorporated 3 drug loadings % of catalase at 5%, 10%, and 20% and measured the catalase activity by enzymatic assay to identify active drug in the BCNPs.

Results: We evaluated surface functionalization strategies in BC pellicle aiming to create functionalized nanoparticles, and benchmarked our methods against literature reported reactions where alpha cellulose was used as a validation for those reactions. Compared to alpha cellulose post-surface modifications, the BC pellicle was positively charged upon amination, while methylation, acetylation, and esterification increased the negative charge of BC, as represented in FTIR and XRD data. The surface chemistry consisted of reactions with the primary and secondary alcohol groups on the BC surface, where the surface chemistry dictated interfacial properties and interactions with other species. Double emulsion formulation allowed the development of BCNPs <100 nm in diameter, slightly negative zeta-potential, and have low PDI post surface modification of BC pellicle. These physicochemical properties align with our prior work for polymer nanoparticles that show promise for delivery of proteins to the brain. The surface modified BCNPs were compared to the chitosan, a positively charged polysaccharide, and alginate as controls for colloidal stability and protein interactions for potential drug loading applications. The biocompatibility results of BCNPs in ex vivo models show BCNPs are non-toxic at lower doses and do not initiate an inflammatory response in healthy brain slices. We also demonstrate that catalase, a low cost and readily available protein, can be incorporated onto surface modified BCNPs by utilizing positively charged or aminated BCNPs that facilitate electronic interactions with the available carbonyls on catalase. Other interactions that could occur between the BCNPs and catalase could potentially be hydrogen bonding stabilizing the platform based on the crystallinity and organization of the cellulose units observed by XRD.

Conclusion:

We formulated sub-100nm BCNPs with positive and negatively charged chemical modifications and benchmarked their protein loading and cytotoxicity in the brain against field standard alginate and chitosan nanoparticles. Our results establish a tunable and sustainably derived platform in nanomedicine for the application of protein-based delivery to the brain. The small library of functionalized BC can open opportunities for other utilizations of BCNPs and an eco-friendlier way to produce therapeutics using green engineering.

References:

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