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Chapter 5

Concluding Remarks

Novel strategies to extend the utilization of bio-based polysaccharide nanofibers in

biomedical materials were successfully built via Pickering emulsion (PE) templating.

Considering the influences of morphology, surface charge and functional groups, native

cellulose nanofiber (CNF), TEMPO-oxidized CNF (TOCNF), chitosan nanofiber (CsNF),

chitin nanofiber (CtNF), phosphorylated CNF (PCNF) and sulfated CNF (SCNF) were applied

as Pickering stabilizers, and their stabilization mechanism was described by controlling solid

content of nanofibers, oil phase ratios and temperatures. Depending on the future application

of these PEs, this dissertation can be roughly divided into three studies in Figure 5.1: 1)

chapter 2 that exploited the usage of 3D cell culture scaffolds based on PEs stabilized with

TOCNF and CsNF via freeze-drying to enhance the enzymatic responses of HepG2 cells; 2)

chapter 3 that investigated the differential cell death behavior induced by TOCNF-stabilized

PE, CtNF-stabilized, β-tricalcuim phosphate nanoparticle-stabilized PE and surfactantstabilized emulsion (SSE) in NIH/3T3, HepG2 and KUP5 cells; 3) chapter 4 that constructed

a stable SCNF-stabilized PE which induced differential inflammatory responses in a cell-type

dependent behavior with high loading capacity of ovalbumin as an antigen. The focal point in

this dissertation is to advance the application of these bio-based polysaccharide nanofibers in

regenerative medicine and immunomodulatory system via a more green and sustainable way.

171

Figure 5.1. Summary illustration of this dissertation.

In chapter 2, a highly porous 3D scaffold was successfully constructed using TOCNF and

CsNF by PE templating method without using any cross-linking agents. Freeze-drying as a

facile and low-cost technique was applied to remove an oil phase. Considering the opposite

surface charge of TOCNF and CsNF, a two-step emulsification method was investigated to

obtain stable PEs with droplet sizes varying from 2 to 10 μm, which was tunable by adjusting

oil phase ratios. The longer CsNFs were located between oil droplets, while the shorter

TOCNF adsorbed to the oil–water interface; both of them contributed to the superior stability

of the PE system. To enhance the emulsion stability, 50 mM NaCl was added into nanofiber

172

suspension before emulsification, and further contributed to improve the porosity of the final

product. The 3D scaffolds demonstrated non-cytotoxicity, and provided suitable

microenvironment for NIH/3T3 and HepG2 cells to proliferate and form spheroids inside of

the scaffolds. Owing to the functional glyco-endowed biointerfaces of the porous scaffolds,

HepG2 spheroids formed inside of the scaffolds exhibited 10-fold higher enzymatic responses

for detoxification metabolism than that cultured on 2D substrates. The obtained results

provided new insights into constructing novel 3D scaffolds via combination of PE templating

and lyophilization. More importantly, it would be promising to further modulate the growth

speed and rate of ice crystals to regulate the pore and aligned structure.

Nano- and microparticles have received extensive attention in designing novel drug

delivery systems and vaccine adjuvants. However, in chapter 2, limited cellular uptake

behavior could be observed due to the utilization of scaffolds. Hence, in chapters 3 and 4, the

cellular uptake and inflammatory responses of polysaccharide nanofiber-stabilized PE

microparticles were explored by using NIH/3T3, HepG2 and KUP5 cells. In chapter 3,

compared with conventional inorganic nanoparticle-stabilized PE and SSE, polysaccharide

nanofiber-stabilized PE induced pyroptototic cell death in HepG2 and KUP5 cells in a dosedependent and cell-type dependent behavior. These PE microparticles were endocytosed by

the cells and resulted in cell swelling, blebbing, caspase-1 activation and interleukin-1β release,

which provided new insights into regulating inflammation-related diseases for developing

novel anticancer drugs and vaccine adjuvants. To further investigate the influence of the

surface functional groups of nanofibers, CNF, PCNF, SCNF and TOCNF were selected to

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prepare PEs, while SPE exhibited the highest physical stability up to two months with limited

droplet size variation. The cellular uptake of these PE microparticles induced the reactive

oxygen species production and lysosome damage in KUP5 cells. Furthermore, SPE indicated

the highest OVA loading capacity up to 85% owing to the strong electrostatic interaction

between SCNF and OVA. This strategy provided new inspiration to formulate advanced

vaccine adjuvants. It would be more necessary to explore the cellular uptake behavior of PE

microparticles in dendritic cell and in vivo models. Moreover, it would be more promising to

investigate the possible phagocytosis mechanism between microparticles and macrophages in

a hepatocyte-Kupffer cells co-cultured system.

The present study engaged important knowledge to extend the usage of bio-based

polysaccharides from forest and marine resources in an eco-friendly and sustainable approach.

The utilization of cellulose-, chitin-, and chitosan-stabilized PEs will greatly advance tissue

engineering, pharmacotherapy and regenerative medicine in the near future.

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