Chitosan (CS) is a ubiquitous biopolymer and is recognized as a promising biomedical material. Potential medical applications for chitosan are extensive and many have shown impressive results. However, chitosan is clinically only used as a topical hemostatic agent due to an inability to sterilize and depyrogenate chitosan without negatively altering its physical and biological properties with conventional techniques. We hypothesized that non-thermal nitrogen plasma (NtNP) would sterilize and depyrogenate chitosan while preserving its biological properties. Since plasma is a surface treatment, we micronized chitosan using cryo-ball and cryo-jet milling to increase its surface to volume ratio. Cryo-jet milling produced the smallest mean particle size (16.05 μm) and reduced molecular weight 36.5% (MW) and degree of deacetylation (DD) 9.6%. NtNP treatment of the resultant chitosan powder produced a sterile chitosan with endotoxin levels <2.50 EU/g, but further reduced MW 40% and DD 6.3%. Nitrogen content and bioadhesivity were unaltered. We tested biologic functionality in a murine orthotopic bladder cancer model in which a previous study showed chitosan dramatically enhanced anti-tumor properties of interleukin-12 (IL-12). We found that bladder tumors regressed – and mice survived to 90 days – in 88% (n=16) and 92% (n=12) of C57Bl/6 mice treated with unsterile chitosan+IL-12 and plasma-sterilized chitosan+IL-12, respectively, but only 8% (n=12) and 0% (n=3) of mice treated with saline and CS-alone, respectively. All plasma-sterilized chitosan+IL-12 (n=11) and 92% of unsterile chitosan+IL-12 (n=13) treated mice rejected tumors upon re-challenge. These results are equivalent to the previously published chitosan+IL-12 study suggesting NtNP sterilization preserved the biological properties of chitosan. A rat model showed that hemostatic chitosan pads sterilized with electron beam irradiation and NtNP stopped femoral artery and artery/vein bleeding better than pads treated with either treatment alone and equivalent to unsterilized pads. A porcine kidney bleeding model showed that NtNP-sterilized pads had superior hemostatic properties compared to e-beam sterilized pads. No complications associated with implanting the pads were observed 14-weeks post-operatively. Histologic examination demonstrated tissue and inflammatory reactions characteristic of foreign body implants. Knowledge gained in these studies will allow clinical testing of parenteral applications of chitosan, including immediate trials of chitosan+IL-12 in bladder cancer patients.

LLU Discipline



Pathology and Human Anatomy


School of Medicine

First Advisor

Kirsch, Wolff M.

Second Advisor

Baldwin, Duane

Third Advisor

Duerksen-Hughes, Penelope J.

Fourth Advisor

Hudson, Samuel

Fifth Advisor

Kirby, Michael A.

Sixth Advisor

Langridge, William

Degree Name

Doctor of Philosophy (PhD)

Degree Level


Year Degree Awarded


Date (Title Page)




Library of Congress/MESH Subject Headings

Bladder Neoplasms - Drug Therapy; Tumor Cells; Cultured; Chitosan - Therapeutic Use; Hemostatic Techniques; Immunotherapy; Adoptive; Interleukin-12

Subject - Local

Plasma Sterilization; Intravesical Immunotherapy; Bladder Cancer; Hemostatic Efficacy



Page Count


Digital Format


Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.


Loma Linda University Electronic Theses and Dissertations

Collection Website



Loma Linda University. Del E. Webb Memorial Library. University Archives